Molecular Immunology 63 (2015) 268–278

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NKG2D receptor activation of NF-␬B enhances inflammatory cytokine production in murine effector CD8+ T cells Emily Whitman, Amorette Barber ∗ Department of Biological and Environmental Sciences, Longwood University, Chichester Science Center 305A, 201 High Street, Farmville, VA 23909, USA

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Article history: Received 29 May 2014 Received in revised form 10 July 2014 Accepted 14 July 2014 Available online 31 July 2014 Keywords: DAP10 Immunotherapy Costimulation NF-␬B CD8T cell Cytokine

a b s t r a c t To induce strong immune responses, naïve CD8+ T cells require stimulation through the TCR and costimulatory receptors. However, the biological effect of activating costimulatory receptors on effector T cells is still unclear. One costimulatory receptor that is likely to be engaged at the target site is NKG2D. This activating receptor is expressed on human and murine CD8+ T cells with its ligands expressed on the majority of tumor cells and during some infections. In order to determine how activation of costimulatory receptors alters effector CD8+ T cell functions, this study compared the activation of the NF-␬B signaling pathway by two costimulatory receptors, CD28 and NKG2D. Compared to CD28 costimulation, activation of murine effector CD8+ T cells through CD3 and NKG2D receptors enhanced activation of NF-␬B as shown by increased phosphorylation of IKK␣, I␬B␣, and NF-␬B and I␬B␣ degradation. NKG2D costimulation also increased activation, nuclear translocation, and DNA binding of NF-␬B p65/p50 dimers. Activation of the NF-␬B pathway also lead to increased gene expression and secretion of pro-inflammatory cytokines, including IFN␣ and IFN␥, and decreased gene expression and secretion of anti-inflammatory cytokines, including IL-10 and CCL2. Altered NF-␬B activation also increased expression of the effector molecules TNF␣, lymphotoxins ␣ and ␤, and Fas ligand, and increased tumor cell killing through FasL. These data show that compared to CD28 costimulation, activation through the NKG2D receptor leads to the differential activation of the NF-␬B signaling pathway and potentially enhances the anti-tumor and anti-viral functions of effector CD8+ T cells. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Complete activation of naïve CD8+ T cells requires stimulation through the T cell receptor (TCR) and costimulatory receptors. The nature of costimulation often shapes the generation of the effector T cell response by altering cytokine secretion, proliferation, survival and other T cell functions. The ligands for many of these costimulatory molecules are also expressed at sites of infection and tumors. Therefore activation of costimulatory receptors also likely alters the effector CD8+ T cell response and contributes to the development of the immune response to tumors and infections. However the biological consequence of activating costimulatory receptors on effector CD8+ T cells is still not well understood.

Abbreviations: NKG2D, Natural Killer Group 2 D; TCR, T cell receptor; PI3K, phosphatidylinositol-3 kinase; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; LT, lymphotoxin; FasL, fas ligand. ∗ Corresponding author. Tel.: +1 434 395 2726; fax: +1 434 395 2652. E-mail address: [email protected] (A. Barber). http://dx.doi.org/10.1016/j.molimm.2014.07.015 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

Typically, the optimum activation of naïve CD8+ T cells results from stimulation through the CD28 receptor which promotes T cell proliferation and survival through secretion of IL-2 and expression of anti-apoptotic proteins (Watts, 2010). However there are many other costimulatory receptors expressed on effector CD8+ T cells that also play a critical role in shaping T cell function (Capece et al., 2012; Sharpe and Abbas, 2006). One of these costimulatory receptors is NKG2D, which is an activating receptor expressed on NK cells, all human CD8+ T cells, activated murine CD8+ T cells and some CD4+ T cells (Ogasawara and Lanier, 2005; Ullrich et al., 2013). Most healthy cells do not express NKG2D ligands however they are often upregulated during DNA damage and cell stress (Champsaur and Lanier, 2011; Mistry and O’Callaghan, 2007; Ogasawara and Lanier, 2005; Raulet, 2003; Ullrich et al., 2013). These ligands are present on many types of tumor cells and are associated with some autoimmune diseases and infections, thus it is highly likely that the NKG2D receptor is stimulated on activated CD8+ T cells during the effector phase of an immune response. In T cells NKG2D associates with an adaptor protein, DAP10, and activates intracellular signaling pathways to provide a costimulation signal (Groh et al., 2001; Lanier, 2009; Maasho et al.,

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2005; Markiewicz et al., 2005). CD28 and DAP10 both contain a YINM-sequence signaling motif (Lanier, 2009; Maasho et al., 2005; Markiewicz et al., 2005). After receptor stimulation, the tyrosine is phosphorylated and phosphatidylinositol-3 kinase (PI3K) and a Grb2–Vav1 complex are consequently activated which leads to downstream activation of AKT and MAP kinases respectively (Lanier, 2009). One way DAP10 and CD28 proteins differ is that DAP10 lacks additional signaling domains responsible for the binding of other signal transduction molecules including Itk, Tec, and Lck (Upshaw and Leibson, 2006). Previous studies have shown that NKG2D induces similar but not identical effects to CD28 in naive and effector CD8+ T cells, further suggesting that the activation of signaling and resulting gene expression may not be identical between the two receptors (Barber and Sentman, 2011; Ehrlich et al., 2005; Groh et al., 2001; Maasho et al., 2005; Markiewicz et al., 2005; Upshaw and Leibson, 2006). Of particular interest to tumor immunity, NKG2D stimulation in effector CD8+ T cells uniquely decreased the expression and secretion of anti-inflammatory cytokines IL-10, IL-9, IL-13 through activation of the ␤-catenin pathway (Barber and Sentman, 2011). Additionally, expression of NKG2D ligands on tumor cells leads to an increase in CD8+ T cell killing, secretion of proinflammatory cytokines IFN␥ and TNF␣, and development of CD8+ T cell memory responses (Barber and Sentman, 2011; Chu et al., 2013; Hessmann et al., 2011; Rajasekaran et al., 2010; Upshaw and Leibson, 2006; Wensveen et al., 2013; Zloza et al., 2012). It is likely that the differential activation of signal transduction pathways by CD28 and NKG2D receptors changes the gene expression profiles and functions of effector T cells, but how this occurs is still not clear. NF-␬B is a transcription factor that enhances the expression of hundreds of genes including cytokines, adhesion molecules, and anti-apoptotic proteins. Cytokine secretion primarily depends on new protein synthesis, therefore transcription rates and activity of transcription factors, such as NF-␬B, play a key role in their regulation (Blackwell and Christman, 1997). Secretion of proinflammatory cytokines is essential for strong anti-tumor and anti-viral T cell responses, thus it is important to understand how costimulatory receptors alter the expression of these cytokines. In T cells, TCR/CD28 activates multiple intermediate signaling proteins including PI3K and AKT to ultimately activate NF-␬B (Cheng et al., 2011; Kane et al., 2002; Tuosto, 2011). While NKG2D also activates PI3K and AKT, it is not known if NKG2D alters TCR-induced NF-␬B signaling in effector CD8+ T cells. This study investigates the activation of the NF-␬B pathway in murine effector CD8+ T cells stimulated through the NKG2D receptor. We determined how simultaneous signals from the TCR complex and NKG2D altered activation of the NF-␬B signaling pathway leading to changes in the expression and secretion of cytokines and other proteins involved in CD8+ T cell anti-tumor and anti-viral responses. These data suggest that the NKG2D receptor regulates ongoing effector CD8+ T cells responses through activation of NF-␬B and may have implications for immune responses to tumors, infections, and autoimmunity due to the expression of NKG2D ligands in these environments.

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Murine splenocytes were stimulated with concanavalin A (ConA; 1 ␮g/ml) for 18 h in complete RPMI media supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 1 mM sodium pyruvate, 10 mM Hepes, 0.1 mM non-essential amino acids and 50 ␮M 2-mercaptoethanol. Cells were then washed and cultured in 25 IU/ml rhu IL-2 (PeproTech, Rocky Hill, NJ) for an additional 6 days in order to induce effector T cells. On day four of T cell culture, CD8+ T cells were negatively purified by a magnetic cell sorting kit (Miltenyi Biotec Inc. Auburn, CA) using anti-CD4 FITC antibodies (eBioscience, San Diego, CA) according to the manufacturer’s instruction. CD28 and NKG2D expression were evaluated on effector CD8+ T cells by flow cytometry. Cells were incubated with FcR block and cell-surface stained with FITC-conjugated anti-CD28 (clone E18, BioLegend) and APC-conjugated anti-NKG2D (clone CX5, BioLegend) or isotype control antibodies. Cell fluorescence was monitored using an Accuri C6 cytometer (BD BioSciences). To stimulate effector T cells, 96-well flat bottom plates were coated overnight at 4 ◦ C with stimulating antibodies against CD3 (1 ␮g/ml, clone 145-2C11) alone, CD3 in combination with CD28 (0.5 ␮g/ml, clone 37.51), and/or with NKG2D (0.5 ␮g/ml, clone A10) or NKG2D alone. The plates were washed three times with PBS, effector T cells (400,000 cells/well for all assays) were added, and cells were stimulated at 37 ◦ C for various times. Anti-CD3 antibodies were purchased from BioLegend (San Diego, CA) and anti-CD28 and anti-NKG2D antibodies were purchased from eBioscience. For experiments with inhibitors, NF-␬B inhibitors ACHP (10 ␮M, Tocris Bioscience, Ellisville, MO), and caffeic acid phenethyl ester (CAPE, 25 ␮g/ml, Tocris Bioscience) were added to the cells at the time of stimulation (Natarajan et al., 1996; Sanda et al., 2005). All inhibitors were dissolved in DMSO and vehicle control contained equal concentration of DMSO specific for each inhibitor. For stimulation with tumor cells, effector T cells (2 × 105 ) were cultured in triplicate with RMA, RMA-Rae1, or Renca cells (2 × 105 ) in 96-well plates. RMA and RMA-Rae1 cells were kindly provided by Dr. Charles Sentman at Dartmouth Medical School (Lebanon, NH) and Renca cells were purchased from ATCC. Anti-CD3 (1 ␮g/ml, clone 145-2C11) or isotype (Armenian hamster IgG; eBioscience) antibodies were also added to the wells. For some stimulations, NF-␬B inhibitor CAPE (25 ␮g/ml) or DMSO vehicle control was also added to the cultures. 2.2. Real time-polymerase chain reaction Effector T cells were stimulated with plate bound antibodies for 8 h and total RNA was isolated using the SV Total RNA Isolation System (Promega, Madison, WI). Random hexamer primers were used to synthesize cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Pittsburgh, PA). Gene expression of NF-␬B pathway members was analyzed via real-time-PCR. Thermo Scientific Maxima SYBR Green qPCR Master Mix and cDNA (5 ng) was used for each reaction according to manufacturer’s instructions. Primers used for RT-PCR reactions were from the Mouse NF-␬B Primer Library (Real Time Primers, LLC, Elkins Park, PA). 2.3. Enzyme-linked immunosorbent assay

2. Materials and methods 2.1. Stimulation of CD8+ T cells Male C57BL/6 (B6) mice were purchased from Harlan Laboratories (Frederick, MD). Mice were between 7 and 12 weeks of age at the start of each experiment. All animal work was performed in accordance and with approval from Longwood University’s Institutional Animal Use and Care guidelines.

Effector T cells were stimulated with plate bound antibodies or tumor cells for 24 h and cell-free supernatants were assayed for IFN␥, IFN␣, TNF␣, IL-10, CCL2, and FasL by ELISA following manufacturer’s instructions. All ELISAs were purchased from BioLegend except for IFN␣ and FasL ELISAs which were purchased from RayBiotech (Norcross, GA). Total protein and phosphorylation of the NF-␬B signal transduction pathway members was measured in effector T cells stimulated with plate bound antibodies for 0, 5, 20 and 60 min. An NF-␬B

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Pathway Activation Profile In-Cell ELISA was used according to manufacturer’s instructions to test for phospho-NF-␬B p65 (Ser 536), phospho-I␬B␣ (Ser 32/36) and phospho-IKK␣ (Ser 176/180), and an NF-␬B p65 (Total/Phospho) In-Cell ELISA was used to test for total levels of p65 protein (eBioscience). NF-␬B p65 and p50 activation and DNA binding activity was measured in effector T cells stimulated with plate bound antibodies for 60 min. TransAM NF-␬B p65 and p50 transcription factor assay kits were used according to manufacturer’s instructions and 10 ␮g of sample was used per well (Activ Motif, Carlsbad, CA). Whole cell extracts were assayed in addition to nuclear extracts which were prepared using the Nuclear Extract kit according to manufacturer’s instructions (Activ Motif). 2.4. Cytotoxicity assay Lysis of target cells (RMA and RMA-Rae1) was determined by a 4 or 24 h LDH cytotoxicity assay according to manufacturer’s instructions (Thermo Scientific Pierce). Effector T cells (105 ) were added to varying numbers of target cells to obtain effector to target ratios of 1:1, 5:1, and 25:1. To block Fas ligand, T cells were preincubated at 37 ◦ C for 2 h with the anti-FasL antibodies (clone MFL3, 20 ␮g/ml, LEAF-purified, BioLegend) or isotype control antibodies (armenian hamster IgG) prior to addition of target cells. To block NF-␬B, T cells were preincubated at 37 ◦ C for 2 h with NF-␬B inhibitor CAPE (25 ␮g/ml) or DMSO vehicle control prior to addition of target cells. 2.5. Statistical analysis The program R was used to run statistical analysis of the data. Differences between groups were analyzed using an unpaired, twotailed Student’s t-test or an ANOVA with a post-hoc Tukey test when comparing multiple groups. The data was determined to be normally distributed using the Shapiro–Wilk test. Samples were assayed in triplicate and values of P < 0.05 were considered significant. 3. Results It is unclear how the NKG2D receptor alters signaling in effector T cells. Stimulation of NKG2D/DAP10 activates PI3K and AKT which can further influence many downstream signaling pathways, including NF-␬B (Lanier, 2009). To study the activation of NF-␬B, we first used an NF-␬B pathway specific RT-PCR array to analyze the expression of multiple genes controlled by this transcription factor. Stimulation through a combination of CD3 and NKG2D receptors differentially altered the expression of multiple genes in the NF-␬B pathway compared to T cells stimulated through CD3 and CD28 (data not shown). These initial data suggested that the NKG2D receptor uniquely activated the NF-␬B pathway in effector CD8+ T cells. 3.1. Stimulation of the NKG2D receptor enhances phosphorylation and activation of NF-ÄB signal transduction pathway members in CD8+ T cells The NF-␬B family consists of p50, p65 (RelA), c-Rel, p52 and RelB (Cheng et al., 2011; Kane et al., 2002; Tuosto, 2011). Different dimer combinations of the NF-␬B subunits have distinct DNA binding specificities and may serve to activate specific sets of genes. In the majority of cells, NF-␬B subunits exist in an inactive form in the cytoplasm, bound to the inhibitory I␬B proteins. NF-␬B proteins are activated by the IKK complex, which phosphorylates I␬B and targets it for proteasomal degradation. This frees NF-␬B dimers and, after phosphorylation, allows these transcription factors to translocate to the nucleus to induce gene expression. In addition to phosphorylation, proteolytic cleavage of p105 (NF-␬B1) results in

the release of p50, which has DNA-binding activity but no transactivation domain. Thus, heterodimers of p50/p65 are some of the most commonly activated NF-␬B subunits that subsequently translocate to the nucleus to induce expression of target genes (Cheng et al., 2011; Kane et al., 2002; Tuosto, 2011). To measure the activation of the NF-␬B pathway, phosphorylation and total levels of NF-␬B signal transduction pathway members were determined (Fig. 1). Phosphorylation of upstream proteins IKK␣ and I␬B, and of NF-␬B p65 itself was increased in T cells costimulated through the NKG2D or CD28 receptors in comparison to CD3 stimulation alone (Fig. 1). In addition, T cells activated through CD3/NKG2D also showed a modest increase in the activation of NF-␬B-pathway members compared to CD3/CD28 stimulation. I␬B phosphorylation decreased in CD3/NKG2D- and CD3/CD28-stimulated T cells overtime due to the degradation of the I␬B protein after phosphorylation (data not shown). While phosphorylation of p65 increased the most in CD3/NKG2D-stimulated cells, total levels of p65 protein did not change in any of the stimulation groups. To determine if p50 was also activated and if NF-␬B p65/p50 dimers have become active transcription factors, a TransAM assay was used to measure binding of p65 and p50 to the NF-␬B consensus site (5 -GGGACTTTCC-3 ). In whole cell extracts, costimulation with either CD28 or NKG2D increased activation and DNA binding activity of p65 compared to CD3 stimulation alone, with NKG2D stimulation showing the largest increase in activity (Fig. 1B). p50 DNA-binding activity showed a similar pattern of activation, with that largest increase in DNA binding being observed in T cells stimulated through CD3 and NKG2D. This showed that NKG2D costimulation lead to an increased amount of p50 activation, likely through cleavage of p105. Importantly, the presence and DNAbinding activity of p65 and p50 also showed enhanced activity in nuclear extracts of stimulated T cells, again with CD3/NKG2D stimulated cells showing the largest increase in p65 and p50 DNA binding. Overall, these data showed that costimulation through the NKG2D receptor enhanced NF-␬B signaling in effector CD8+ T cells, leading to increased translocation and activity of p65/p50 dimers. 3.2. Activation through the TCR and NKG2D increases proinflammatory cytokine expression Effector CD8+ T cells secrete proinflammatory cytokines to help induce strong immune responses. NF-␬B plays a crucial role in gene regulation of cytokines; therefore, NKG2D-stimulation may alter the gene expression of cytokines through activation of the NF-␬B pathway. Stimulation of effector CD8+ T cells through CD3/NKG2D receptors increased gene expression and secretion of proinflammatory cytokines, IFN␣ and IFN␥, compared to CD3 or CD3/CD28 stimulation (Fig. 2). While the scale of the graph makes the expression levels in CD3-stimulated T cells appear low, it is interesting to note that compared to unstimulated CD8T cells, the gene expression for IFN␣1 is 3.9 fold higher and IFN␣2 is 5.3 fold higher. This shows that CD3-stimulus alone leads to a modest increase in IFN␣ expression, and that CD28 and NKG2D signaling can amplify this expression, with NKG2D having the largest increase in IFN␣ expression. However, activating the NKG2D receptor alone did not increase expression of these cytokines, showing the requirement for TCR signaling. Other proteins that are induced by the NF-␬B pathway are members of the TNF superfamily, TNF␣ and lymphotoxins (LT) ␣ and ␤, all of which have important roles in inducing inflammatory immune responses (Upadhyay and Fu, 2013; Ware, 2005). In CD8+ T cells, CD3/NKG2D stimulation increased gene expression of TNF␣ and LT␣ and ␤ compared to stimulation through CD3/CD28 or CD3 alone (Fig. 3). CD3/NKG2D stimulation also increased the secretion of TNF␣ compared to the other stimuli. Again, T cells stimulated

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Fig. 1. Stimulation of the NKG2D receptor enhances phosphorylation and activation of NF-␬B signal transduction pathway members in CD8+ T cells. Murine effector CD8+ T cells were stimulated with media (filled diamonds), or plate-bound anti-CD3 (filled squares), anti-CD3/CD28 (filled triangles), or anti CD3/NKG2D antibodies (open squares). (A) Phospho-IKK␣ (Ser 176/180), phospho-I␬B␣ (Ser 32/36), phospho-NF-␬B p65 (Ser 536), and total NF-␬B p65 were measured using an In-Cell ELISA. Phosphorylation and total protein was measured at 5, 20 and 60 min. (B) CD8+ T cells were stimulated with antibodies for 60 min and whole cell extracts (filled bars) and nuclear extracts (open bars) were assayed for NF-␬B p65 and p50 activation and DNA binding activity using a TransAM transcription factor assay kit. Phosphorylation or activation was significantly different (* P < 0.05) compared to stimulation through CD3/NKG2D. The data are presented as mean of triplicates plus standard deviation and is one representative of two separate experiments.

through the NKG2D receptor alone did not increase expression of these genes, demonstrating the requirement for concurrent activation of the TCR to induce NF-␬B signaling. 3.3. FasL expression is increased on CD8+ T cells activated through CD3 and NKG2D receptors Another important anti-tumor function of T cells is inducing target cell apoptosis. Fas ligand (FasL) is one protein that induces apoptosis in target cells and can be regulated by the NF-␬B pathway (Kasibhatla et al., 1999). CD3/NKG2D stimulated T cells increased expression of FasL compared to stimulation through CD3/CD28, CD3 alone, or the NKG2D receptor alone (Fig. 4). These data show that the combination of CD3/NKG2D signaling provides the strongest expression of multiple effector functions in effector CD8+ T cells. 3.4. Stimulation through CD3 and NKG2D receptors decreases expression of anti-inflammatory cytokines Anti-inflammatory cytokines such as IL-10 are not often desirable during an immune response against tumors or viruses

because they inhibit the induction of robust inflammatory immune responses. Additionally, CCL2 can recruit anti-inflammatory macrophages to the tumor site thus also inhibiting tumor immunity (Lee et al., 2013). Thus for an optimal response, it would likely be beneficial to inhibit T cell expression of these cytokines. Expression and secretion of IL-10 and CCL2 showed the greatest increase when T cells were stimulated through CD3/CD28 (Fig. 5). Comparatively, CD3/NKG2D stimulation did not induce the expression of these anti-inflammatory cytokines. Therefore, while CD3/NKG2D increased proinflammatory cytokine secretion, it did not concurrently induce anti-inflammatory cytokine secretion unlike CD3/CD28 stimulation.

3.5. NF-ÄB is required for alterations in expression of proinflammatory and anti-inflammatory cytokines To determine if activation of NF-␬B was responsible for altering the expression of effector molecules in CD8+ T cells, the NF␬B inhibitor ACHP, which inhibits IKK␣ and ␤ activation, was added to T cell cultures during receptor stimulation (Sanda et al.,

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Fig. 2. Activation through the TCR and NKG2D increases proinflammatory cytokine expression. Murine effector CD8+ T cells were stimulated with media, or plate-bound anti-CD3, anti-CD3/CD28, anti CD3/NKG2D or anti-NKG2D antibodies. (A) After 8 h, gene expression of proinflammatory cytokines IFN␣1, IFN␣2, and IFN␥ was measured by RT-PCR. Data are shown as the fold change in gene expression compared to T cells stimulated in media alone. (B) After 24 h, secretion of IFN␣ and IFN␥ was measured in cell-free supernatants by ELISA. Cytokine expression was significantly different (* P < 0.05) compared to stimulation through CD3/NKG2D. The data are presented as mean of triplicates plus standard deviation and is one representative of at least two separate experiments.

2005). Inhibition of NF-␬B decreased gene expression of inflammatory cytokines IFN␣, IFN␥, and TNF␣, and the apoptosis-inducing molecule FasL in all stimulated cells, including CD8+ T cells activated through CD3/NKG2D (Fig. 6). In addition, inhibition of NF-␬B also led to the restoration of gene expression of anti-inflammatory cytokines IL-10 and CCL2 in CD3/NKG2D-stimulated cells. A second NF-␬B inhibitor, caffeic acid phenethyl ester (CAPE),which prevents translocation of the p65 subunit of NF-␬B to the nucleus, had similar effects in altering gene expression in stimulated T cells (Fig. 6B) (Natarajan et al., 1996). This suggests that the increased activation of the p65/p50 NF-␬B dimers observed in NKG2D-stimulated T cells contributed to the enhanced cytokine secretion seen in NKG2Dstimulated T cells. 3.6. Stimulation of CD8 T cells with NKG2D-ligand positive tumor cells alters cytokine secretion and tumor cell lysis in an NF-ÄB-dependent manner To evaluate the signaling capacity of NKG2D in response to a more physiologically relevant stimulation, effector CD8T cells were stimulated with murine tumor cells that were NKG2D-ligand negative (RMA cells), or NKG2D-ligand positive (RMA-Rae1 cells which have been transduced to express Rae1 and Renca cells which

naturally expresses NKG2D ligands) (Mistry and O’Callaghan, 2007; Raulet, 2003; Ullrich et al., 2013; Zhang et al., 2007). Anti-CD3 antibodies were also added the cultures to trigger TCR signaling. In the presence of anti-CD3 antibodies and NKG2D ligand-positive tumor cells, CD8T cells secreted more proinflammatory cytokines than CD3 stimulation alone (Fig. 7). In addition, CD3 stimulation alone induced secretion of anti-inflammatory cytokines IL-10 and CCL2, whereas stimulation by NKG2D ligand positive tumor cells inhibited secretion of these cytokines. This NKG2D-driven alteration in cytokine secretion was also dependent on p65/p50 NF-␬B signaling because addition of NF-␬B inhibitor CAPE to the T cell cultures prevented the secretion of proinflammatory cytokines and the decrease in anti-inflammatory cytokines. CD8T cells were the main source of these cytokines in these cultures because tumor cells alone did not produce a significant amount of cytokines, and were not affected by CAPE treatment. Therefore, the NF-␬B dependent alteration in cytokine secretion seen with the previously described antibody-crosslinking studies also occurred when CD8T cells were stimulated by tumor cells expressing NKG2D ligands. In addition to altering cytokine secretion, NKG2D stimulation also increased FasL expression on effector CD8T cells. To determine if this change in FasL expression increased tumor cell killing, cytotoxicity assays were performed against RMA and RMA-Rae1 cells.

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Fig. 3. CD8+ T cells increase expression of TNF␣ family members after activation through CD3 and NKG2D. Murine effector CD8+ T cells were stimulated with media, or plate-bound anti-CD3, anti-CD3/CD28, anti CD3/NKG2D or anti-NKG2D antibodies. (A) After 8 h, gene expression of TNF␣ and LT␣ and ␤ was measured by RT-PCR. Data are shown as the fold change in gene expression compared to T cells stimulated in media alone. (B) After 24 h, secretion of TNF␣ was measured in cell-free supernatants by ELISA. Cytokine expression was significantly different (* P < 0.05) compared to stimulation through CD3/NKG2D. The data are presented as mean of triplicates plus standard deviation and is one representative of two to three separate experiments.

While addition of CD3 stimulation increased killing of both NKG2D ligand positive and negative cells by effector CD8T cells, the presence of NKG2D ligands on the tumor cells further increased T cell killing (Fig. 7B). The increased cytotoxic ability of T cells was dependent on NF-␬B signaling because blocking p65/p50 dimers with the CAPE inhibitor prevented the increase in killing seen in the presence of NKG2D ligands. Furthermore, this increase in cytotoxicity was also dependent on Fas ligand, because the increased killing ability was lost when Fas ligand was blocked on the T cells. Tumor cell killing was only partially inhibited when blocking Fas ligand because RMA cells are also susceptible to killing by perforin (Zhang et al., 2007). These data also support similar studies showing that T cells expressing a chimeric NKG2D receptor (consisting of the NKG2D receptor fused to the CD3␨ chain) killed RMA-Rae1 cells but not RMA cells in a partially FasL-dependent manner in vitro and in vivo (Zhang et al., 2005, 2007). Together, these data show that the increased activation of p65/p50 NF-␬B dimers by the NKG2D receptor enhanced tumor cell killing through increased Fas ligand expression. 3.7. Costimulation of T cells through NKG2D modulates CD3/CD28 signal

Fig. 4. FasL expression is increased on CD8+ T cells activated through CD3 and NKG2D receptors. Murine effector CD8+ T cells were stimulated with media, or plate-bound anti-CD3, anti-CD3/CD28, anti CD3/NKG2D or anti-NKG2D antibodies. (A) After 8 h, gene expression of FasL was measured by RT-PCR. Data are shown as the fold change in gene expression compared to T cells stimulated in media alone. (B) After 24 h, secretion of FasL was measured in cell-free supernatants by ELISA. Cytokine expression was significantly different (* P < 0.05) compared to stimulation through CD3/NKG2D. The data are presented as mean of triplicates plus standard deviation and is one representative of two to three separate experiments.

At the site of a tumor or infection, it is likely that multiple costimulatory receptors may be triggered on the surface of a CD8+ T cell. To first determine if NKG2D and CD28 signaling might occur on the same cell, the expression of these receptors on effector CD8T cells was analyzed (Fig. 8). Both receptors were expressed on the same population of T cells, indicating that it is possible that these receptors could be simultaneously activated on the same cell. To test the effect of signaling through the TCR, NKG2D, and CD28 receptors simultaneously, T cells were activated with a combination of anti-CD3, anti-NKG2D, and anti-CD28 antibodies. Concurrent costimulation of NKG2D and CD28 receptors modulated the activation of p65 and p50 and showed an intermediate activation of these

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Fig. 5. Stimulation through CD3 and NKG2D receptors decreases expression of anti-inflammatory cytokines. Murine effector CD8+ T cells were stimulated with media, or plate-bound anti-CD3, anti-CD3/CD28, anti CD3/NKG2D or anti-NKG2D antibodies. (A) After 8 h, gene expression of anti-inflammatory cytokines IL-10 and CCL2 was measured by RT-PCR. Data are shown as the fold change in gene expression compared to T cells stimulated in media alone. (B) After 24 h, secretion of IL-10 and CCL2 was measured in cell-free supernatants by ELISA. Cytokine expression was significantly different (* P < 0.05) compared to stimulation through CD3/NKG2D. The data are presented as mean of triplicates plus standard deviation and is one representative of two to three separate experiments.

Fig. 6. NF-␬B is required for alterations in expression of proinflammatory and anti-inflammatory cytokines. Murine effector CD8+ T cells were stimulated with media, or plate-bound anti-CD3 (X), anti-CD3/CD28 (open triangles), anti CD3/NKG2D (open diamonds) or anti-NKG2D (filled diamonds) antibodies in the presence of the (A) NF-␬B inhibitor ACHP (10 ␮M), or DMSO vehicle control (0 ␮M) or (B) NF-␬B inhibitor CAPE (25 ␮g/ml), or DMSO vehicle control (0 ␮g/ml) for 8 h. Gene expression was determined by RT-PCR. Data are shown as the fold change in gene expression compared to T cells cultured in media at each concentration of inhibitor. The data are presented as mean of triplicates plus standard deviation and is one representative of two experiments.

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Fig. 7. Stimulation of CD8T cells with NKG2D-ligand positive tumor cells alters cytokine secretion and tumor cell lysis in an NF-␬B-dependent manner. (A) Murine effector CD8+ T cells were stimulated with RMA, RMA-Rae1, or Renca tumor cells in the presence of DMSO vehicle control and anti-CD3 (black bars) or isotype control antibodies (white bars) or NF-␬B inhibitor CAPE (25 ␮g/ml) and anti-CD3 (dark grey bars) or isotype control antibodies (light grey bars). Tumor cells were also cultured in media alone with DMSO (vertical striped bars) or CAPE (horizontal striped bars). After 24 h, secretion of cytokines and FasL was measured in cell-free supernatants by ELISA. Cytokine expression was significantly different (* P < 0.05) compared to stimulation through RMA cells. (B) Lysis of RMA cells or RMA-Rae1 cells by effector CD8T cells was measured at the indicated E:T ratios (1:1, 5:1, 25:1) after 24 h. In the left panels, T cells were stimulated with either anti-CD3 (circles) or isotype control antibodies (squares) in the presence of the NF-␬B inhibitor CAPE (open symbols) or DMSO vehicle control (closed symbols). In the right panels, T cells were stimulated with either anti-CD3 (circles) or isotype control antibodies (squares) in the presence of blocking anti-FasL antibodies (open symbols) or isotype control antibodies (closed symbols). Inhibiting NF-␬B or FasL significantly decreased specific lysis of tumor cells (* P < 0.05). The data are presented as mean of triplicates plus standard deviation and is one representative of two separate experiments. Similar lysis data was obtained after 4 h of incubation.

proteins, compared to NKG2D or CD28 costimulation alone (Fig. 8). The combination of signaling through these two receptors also led to an increase in IFN␣, IFN␥, TNF␣, and FasL secretion and a decrease in IL-10 and CCL2 compared to cells stimulated through CD3/CD28. Together, these data show that activation of the NKG2D receptor on effector T cells altered CD28 signaling by activating NF-␬B and resulted in an increased secretion of proinflammatory cytokines and a decreased expression of anti-inflammatory cytokines. 4. Discussion While complete activation of naïve CD8+ T cells requires stimulation through the TCR and costimulatory receptors, continued activation of costimulatory receptors at the target site likely alters CD8+ T cell effector and memory responses to shape the immune response to tumors and infections. The data in this study show that activation of the NKG2D receptor in conjunction with the TCR increases the activation of p65/p50 NF-␬B dimers and downstream genes in murine effector CD8+ T cells. NKG2D-induced activation

of NF-␬B increased the secretion of proinflammatory cytokines and decreased anti-inflammatory cytokines compared to T cells stimulated through CD3 and CD28 or CD3 alone. NKG2D stimulation also enhanced killing of NKG2D-ligand positive tumor cells due to increased Fas ligand expression. Thus, activation of different costimulatory receptors uniquely alters the functions of effector CD8+ T cells. In CD8+ T cells, the NKG2D receptor induces signaling via the associated adaptor molecule DAP10. Previous studies have shown that in human naïve CD8+ T cells, costimulation with NKG2D was similar to CD28 and was able to increase naïve T cell proliferation, cytotoxicity, and production of proinflammatory cytokines IFN␥, TNF␣, and IL-2 (Barber and Sentman, 2011; Ehrlich et al., 2005; Groh et al., 2001; Lanier, 2009; Maasho et al., 2005; Markiewicz et al., 2005; Upshaw and Leibson, 2006). This study compared the costimulation of NKG2D and CD28 in effector CD8+ T cells. Both costimulatory receptors activated NF-␬B, however NKG2D stimulation differentially regulated genes downstream of NF-␬B to ultimately alter effector CD8+ T cell functions. While both NKG2D and CD28 activate PI3K and AKT, recruitment of additional

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Fig. 8. Costimulation of T cells through NKG2D modulates CD3/CD28 signal. (A) Cell surface CD28 and NKG2D receptor expression was determined on murine effector CD8+ T cells by flow cytometry. A representative dot plot is shown. (B) Murine effector CD8+ T cells were stimulated with media, or plate-bound anti-CD3, anti-CD3/CD28, anti-CD3/NKG2D, anti-NKG2D, or anti-CD3/CD28/NKG2D antibodies for 60 min and whole cell extracts (filled bars) and nuclear extracts (open bars) were assayed for NF-␬B p65 and p50 activation and DNA binding activity using a TransAM transcription factor assay kit. (C) After 24 h, secretion of IFN␣, IFN␥, TNF␣, FasL, IL-10, and CCL2 was measured in cell-free supernatants of stimulated CD8+ T cells by ELISA. NF-␬B activation or cytokine secretion was significantly different (* P < 0.05) compared to stimulation through CD3/NKG2D. The data are presented as mean of triplicates plus standard deviation and is one representative of two separate experiments.

signaling molecules to the intracellular domain of CD28 may activate alternative proteins and modulate different signal cascades (Lanier, 2009; Upshaw and Leibson, 2006). Previous studies have shown that NKG2D signaling in effector T cells activated ␤-catenin and downstream genes whereas CD28 did not activate this pathway (Barber and Sentman, 2011). Thus, in conjunction with these data, it is clear that NKG2D and CD28 differently activate various signal transduction cascades and effector responses in activated CD8+ T cells. Most costimulatory molecules, including CD28, ICOS, 4-1BB, CD27, and OX-40, activate NF-␬B, thus emphasizing the importance of this pathway for T cell function (Kane et al., 2002). Nuclear translocation of NF-␬B transcription factors is controlled by the activation of IKK and subsequent degradation of I␬B proteins. Many studies have shown the CD28 costimulation activates AKT which subsequently increases IKK activity and long-term downregulation of I␬B (Harhaj et al., 1996; Kane et al., 2002; Lai and Tan, 1994). Intriguingly, CD28 activation alone is unable to activate NF␬B in T cells, and concurrent TCR signaling is required to activate the IKK complex, induce NF-␬B nuclear translocation, and increase cytokine expression (Kane et al., 2001, 1999). Our data also shows that TCR stimulation is required for NF-␬B activation since T cells stimulated through NKG2D alone did not activate NF-␬B. NKG2D differentially activated p65/p50 NF-␬B dimers compared to CD28

and led to increased nuclear translocation and DNA-binding activity. It would be interesting to further study how CD28 and NKG2D costimulation activate other NF-␬B signal transduction pathway members. Similar to CD4+ T cells, CD8+ T cells can be classified into effector subsets, with Tc1 cells secreting proinflammatory cytokines such as IFN␥ and TNF␣, and Tc2 cells secreting Th2- associated cytokines IL-4, IL-9, and IL-13 (Miyahara et al., 2004; Shrikant et al., 2010). Effector CD8+ T cells can also decrease inflammatory responses through IL-10 secretion (Barber and Sentman, 2011; Endharti et al., 2005; Stumhofer et al., 2007; Sun et al., 2009). However, the signals that control the development of these CD8+ T cell subsets are still unclear. Previous work showed that NKG2D activation on human effector CD8+ T cells decreased expression of anti-inflammatory cytokines IL-10, IL-9, and IL-13 in a ␤-catenin and PPAR␥ dependent manner and increased expression of inflammatory cytokines IFN␥ and TNF␣ in a ␤-catenin and PPAR␥ independent manner (Barber and Sentman, 2011). In comparison, these new data showed that NKG2D activation inhibited IL-10 and CCL2 expression and increased IFN␥, IFN␣, and TNF␣ expression in an NF-␬B-dependent manner. While the regulation of proinflammatory cytokines seems to be solely dependent on NF-␬B in murine CD8+ T cells, it is interesting that IL-10 expression was controlled by NF-␬B and ␤-catenin (Barber and Sentman, 2011). It is still unclear how NKG2D inhibits

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cytokine expression, and future studies should focus on studying how NKG2D signaling inhibits the expression of IL-10 and CCL2. One potential mechanism may be through the concurrent activation of PPAR␥, a transcription factor downstream of ␤-catenin, which can either repress or activate the expression of genes, including IL-10 (Clark, 2002; Deng et al., 2004; Du and Gellar, 2010; Straus and Glass, 2007). NF-␬B and PPAR␥ have recently been shown to co-repress target gene expression, suggesting that a similar mechanism may be occurring to inhibit IL-10 and CCL2 expression in NKG2D-stimulated cells (Gurevich et al., 2012). Future studies should further investigate the intersection of these two pathways in effector CD8+ T cells to determine how they interact to regulate cytokine secretion. Another interesting finding is that NKG2D stimulation greatly increased secretion of the cytokine IFN␣. IFN␣ has been shown to have opposing effects on CD8+ T cells, being able to increase proliferation or decrease proliferation and induce apoptosis through STAT1 activation (Welsh et al., 2012). However activated CD8+ T cells downregulate STAT1, thus allowing for the activation of other pathways that inhibit apoptosis and promote proliferation (Welsh et al., 2012). Although other transcription factors such as IRFs and c-Jun can associate with coactivators, the NF-␬B p65 subunit seems to be unique in its ability to synergize with coactivators and other enhanceosome components controlling the expression of type I interferon genes (Balachandran and Beg, 2011; Merika et al., 1998). Thus, it is possible that in NKG2D-stimulated T cells, the increased activation and nuclear translocation of p65/p50 dimers allows for enhanced transcription of IFN␣. While CD8+ T cells are not usually recognized for their secretion of IFN␣, NKG2D induction of this cytokine may support the maintenance of effector CD8+ T cell responses through induction of T cell proliferation at the tumor or infection site. In addition to regulating cytokines, NKG2D-induced activation of NF-␬B increased the expression of other proteins essential for strong anti-tumor and anti-viral immune responses. Fas ligand and lymphotoxins ␣ and ␤ had a similar expression pattern to the proinflammatory cytokines, with CD3/NKG2D stimulation leading to a stronger induction of these proteins compared to stimulation with CD3/CD28 or CD3 alone. FasL induces apoptosis in target cells and has been shown to often play a critical role in anti-tumor and anti-viral immunity. NF-␬B is a positive regulator of FasL expression thus supporting our finding that NKG2D-induced activation of NF-␬B increased FasL expression and enhanced tumor cell killing (Kasibhatla et al., 1999). Lymphotoxins ␣ and ␤ are members of the TNF superfamily and have been found to mediate inflammation and target cell lysis, and to play a role in the defense against viruses and tumors (Upadhyay and Fu, 2013; Ware, 2005). Interestingly, coexpression of FasL and LT␣ on effector CD8+ T cells can induce strong anti-tumor responses, thus the NKG2D-induced increase of these proteins will likely contribute to enhanced immunity (Dobrzanski et al., 2004). The ligands for the NKG2D receptor are upregulated during DNA damage or cell stress and are expressed on many tumors and after infection with some pathogens, including HIV, influenza virus, and Mycobacterium tuberculosis (Champsaur and Lanier, 2011; Ullrich et al., 2013). While the ligands are not expressed on most healthy cells, deregulated expression of these ligands has been implicated in the development of autoimmune diseases including Crohn’s and celiac diseases and Type I diabetes (Champsaur and Lanier, 2011; Ullrich et al., 2013). These data suggest that NKG2D-induced changes in cytokine secretion may be one mechanism that regulates CD8+ T cell activity. When effector CD8+ T cells encounter NKG2D ligands on tumor or infected cells, NKG2D stimulation may skew CD8+ T cells toward secretion of proinflammatory cytokines and apoptosis-inducing molecules while inhibiting anti-inflammatory cytokine production. This would

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drive the development of a strong inflammatory immune response to efficiently eliminate the pathogen or tumor. As the infection or tumor is cleared, NKG2D ligand expression will decrease, and may prevent a damaging inflammatory response by switching the cytokine profile toward an anti-inflammatory state. However, chronic infection or deregulated NKG2D ligand expression may result in continual NKG2D activation and could possibly lead to long-term tissue damage and development of autoimmunity. In addition to learning about the signals controlling T cell activation and immune responses, studying the effect of costimulation is also of great importance because these receptors are current targets for cancer and anti-viral immunotherapies (Capece et al., 2012; Sharpe and Abbas, 2006). In summary, these data show that NKG2D-costimulation of murine effector CD8+ T cells activated the p65/p50 NF-␬B pathway, and led to an increased expression of proinflammatory cytokines, induction of tumor cell apoptosis, and a decrease in anti-inflammatory cytokines. This activation profile was unique compared to CD28 costimulation. Therefore, activation of the NKG2D receptor and the NF-␬B pathway may induce stronger effector functions in CD8+ T cells. In addition, stimulation of NKG2D on activated T cells may have beneficial effects through reducing the expression of anti-inflammatory cytokines and thus promoting the development of immune responses to infections or cancer. Conflict of interest disclosure The authors declare no conflict of interest. Acknowledgements This work was supported in part by Longwood University’s Faculty Research Grants, PRISM program, and Department of Biological and Environmental Sciences. References Balachandran, S., Beg, A.A., 2011. Defining emerging roles for NF-␬B in antivirus responses: revisiting the interferon-␤ enhanceosome paradigm. PLoS Pathog. 7, e1002165. Barber, A., Sentman, C.L., 2011. NKG2D receptor regulates human effector T-cell cytokine production. Blood 117, 6571–6581. Blackwell, T.S., Christman, J.W., 1997. The role of nuclear factor-␬B in cytokine gene regulation. Am. J. Respir. 17, 3–9. Capece, D., Verzella, D., Fischietti, M., Zazzeroni, F., Alesse, E., 2012. Targeting costimulatory molecules to improve antitumor immunity. J. Biomed. Biotechnol. 2012, e926321. Champsaur, M., Lanier, L.L., 2011. Effect of NKG2D ligand expression on host immune responses. Immunol. Rev. 235, 267–285. Cheng, J., Montecalvo, A., Kane, L.P., 2011. Regulation of NF-␬B induction by TCR/CD28. Immunol. Res. 50, 113–117. Chu, T., Tyznik, A.J., Roepke, S., Berkley, A.M., Woodward-Davis, A., Pattacini, L., Bevan, M.J., Zehn, D., Prlic, M., 2013. Bystander-activated memory CD8 T cells control early pathogen load in an innate-like, NKG2D-dependent manner. Cell Rep. 3, 701–708. Clark, R.B., 2002. The role of PPARs in inflammation and immunity. J. Leukoc. Biol. 71, 388–400. Deng, J., Xia, W., Miller, S.A., Wen, Y., Wang, H.Y., Hung, M.C., 2004. Crossregulation of NF-kappaB by the APC/GSK-3beta/beta-catenin pathway. Mol. Carcinog. 39, 139–146. Dobrzanski, M.J., Reome, J.B., Hollenbaugh, J.A., Hylind, J.C., Dutton, R.W., 2004. Effector cell-derived lymphotoxin alpha and Fas ligand, but not perforin, promote Tc1 and Tc2 effector cell-mediated tumor therapy in established pulmonary metastases. Cancer Res. 64, 406–414. Du, Q., Gellar, D.A., 2010. Cross-regulation between Wnt and NF-␬B signaling pathways. Immunopathol. Dis. Ther. 1, 155–181. Ehrlich, L.I., Ogasawara, K., Hamerman, J.A., Takaki, R., Zingoni, A., Allison, J.P., Lanier, L.L., 2005. Engagement of NKG2D by cognate ligand or antibody alone is insufficient to mediate costimulation of human and mouse CD8+ T cells. J. Immunol. 174, 1922–1931. Endharti, A.T., Rifa, I.M., Shi, Z., 2005. Cutting edge: CD8+ CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells. J. Immunol. 175, 7093–7097.

278

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Groh, V., Rhinehart, R., Randolph-Habecker, J., Topp, M.S., Riddell, S.R., Spies, T., 2001. Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2, 255–260. Gurevich, I., Zhang, C., Encarnacao, P.C., Struzynski, C.P., Livings, S.E., Aneskievich, B.J., 2012. PPAR␥ and NF-␬B regulate the gene promoter activity of their shared repressor, TNIP1. Biochim. Biophys. Acta 1819, 1–15. Harhaj, E.W., Maggirwar, S.B., Good, L., Sun, S.C., 1996. CD28 mediates a potent costimulatory signal for rapid degradation of I␬B␤ which is associated with accelerated activation of various NF-␬B/Rel heterodimers. Mol. Cell. Biol. 16, 6736–6743. Hessmann, M., Rausch, A., Rückerl, D., Adams, P.S., Simon, M., Gilfillan, S., Colonna, M., Ehlers, S., Hölscher, C., 2011. DAP10 contributes to CD8(+) T cell-mediated cytotoxic effector mechanisms during Mycobacterium tuberculosis infection. Immunobiology 216, 639–647. Kane, L.P., Lin, J., Weiss, A., 2002. It’s all Rel-ative: NF-kappaB and CD28 costimulation of T-cell activation. Trends Immunol. 23, 413–420. Kane, L.P., Andres, P.G., Howland, K.C., Abbas, A.K., Weiss, A., 2001. Akt provides the CD28 costimulatory signal for upregulation of IL-2 and IFN-␥ but not Th2 cytokines. Nat. Immunol. 2, 37–44. Kane, L.P., Shapiro, V.S., Stokoe, D., Weiss, A., 1999. Induction of NF-␬B by the Akt/PKB kinase. Curr. Biol. 9, 601–604. Kasibhatla, S., Genestier, L., Green, D.R., 1999. Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor kappaB. J. Biol. Chem. 274, 987–992. Lai, J.-H., Tan, T.-H., 1994. CD28 signaling causes a sustained down-regulation of I␬B␣ which can be prevented by the immunosuppressant rapamycin. J. Biol. Chem. 269, 30077–30080. Lanier, L.L., 2009. DAP10-and DAP12-associated receptors in innate immunity. Immunol. Rev. 227, 150–160. Lee, H.W., Choi, H.J., Ha, S.J., Lee, K.T., Kwon, Y.G., 2013. Recruitment of monocytes/macrophages in different tumor microenvironments. Biochim. Biophys. Acta 1835, 170–179. Maasho, K., Opoku-Anane, J., Marusina, A.I., Coligan, J.E., Borrego, F., 2005. NKG2D is a costimulatory receptor for human naive CD8+ T cells. J. Immunol. 174, 4480–4484. Markiewicz, M.A., Carayannopoulos, L.N., Naidenko, O.V., Matsui, K., Burack, W.R., Wise, E.L., Fremont, D.H., Allen, P.M., Yokoyama, W.M., Colonna, M., Shaw, A.S., 2005. Costimulation through NKG2D enhances murine CD8+ CTL function: similarities and differences between NKG2D and CD28 costimulation. J. Immunol. 175, 2825–2833. Merika, M., Williams, A.J., Chen, G., Collins, T., Thanos, D., 1998. Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol. Cell 1, 277–287. Mistry, A.R., O’Callaghan, C.A., 2007. Regulation of ligands for the activating receptor NKG2D. Immunology 121, 439–447. Miyahara, N., Swanson, B.J., Takeda, K., Taube, C., Miyahara, S., Kodama, T., Dakhama, A., Ott, V.L., Gelfand, E.W., 2004. Effector CD8+ T cells mediate inflammation and airway hyper-responsiveness. Nat. Med. 10, 865–869. Natarajan, K., Singh, S., Burke Jr., T.R., Grunberger, D., Aggarwal, B.B., 1996. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proc. Nat. Acad. Sci. U.S.A. 93, 9090– 9095.

Ogasawara, K., Lanier, L.L., 2005. NKG2D in NK and T cell-mediated immunity. J. Clin. Immunol. 25, 534–540. Rajasekaran, K., Xiong, V., Fong, L., Gorski, J., Malarkannan, S., 2010. Functional dichotomy between NKG2D and CD28-mediated co-stimulation in human CD8+ T cells. PLoS One 5, e12635. Raulet, D.H., 2003. Roles of NKG2D immunoreceptor and its ligands. Nat. Rev. Immunol. 3, 781–790. Sanda, T., Iida, S., Ogura, H., Asamitsu, K., Murata, T., Bacon, K.B., Ueda, R., Okamoto, T., 2005. Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor. Clin. Cancer Res. 11, 1974–1982. Sharpe, A., Abbas, A., 2006. T-cell costimulation—biology, therapeutic potential, and challenges. N. Engl. J. Med. 355, 973–975. Shrikant, P.A., Rao, R., Li, Q., Kesterson, J., Eppolito, C., Mischo, A., Singhal, P., 2010. Regulating functional cell fates in CD8 T cells. Immunol. Res. 46, 12–22. Straus, D.S., Glass, C.K., 2007. Anti-inflammatory actions of PPAR ligands: new insights on cellular and molecular mechanisms. Trends Immunol. 28, 551–558. Stumhofer, J.S., Silver, J.S., Laurence, A., Porrett, P.M., Harris, T.H., Turka, L.A., Ernst, M., Saris, C.J., O’Shea, J.J., Hunter, C.A., 2007. Interleukins 27 and 6 induce STAT3mediated T cell production of interleukin 10. Nat. Immunol. 8, 1363–1371. Sun, J., Madan, R., Karp, C.L., Braciale, T.J., 2009. Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10. Nat. Med. 15, 277–284. Tuosto, L., 2011. NF-␬B family of transcription factors: biochemical players of CD28 co-stimulation. Immunol. Lett. 135, 1–9. Ullrich, E., Koch, J., Cerwenka, A., Steinle, A., 2013. New prospects on the NKG2D/NKG2DL system for oncology. Oncoimmunology 2, e26097. Upadhyay, V., Fu, Y.X., 2013. Lymphotoxin signalling in immune homeostasis and the control of microorganisms. Nat. Rev. Immunol. 13, 270–279. Upshaw, J.L., Leibson, P.J., 2006. NKG2D-mediated activation of cytotoxic lymphocytes: unique signaling pathways and distinct functional outcomes. Semin. Immunol. 18, 167–175. Ware, C.F., 2005. Network communications: lymphotoxins, LIGHT, and TNF. Annu. Rev. Immunol. 23, 787–819. Watts, T., 2010. Staying alive: T cell costimulation, CD28, and Bcl-xL. J. Immunol. 185, 3785–3787. Welsh, R.M., Bahl, K., Marshall, H.D., Urban, S.L., 2012. Type 1 interferons and antiviral CD8 T-cell responses. PLoS Pathog. 8, e1002352. Wensveen, F.M., Lenartic, M., Jelencic, V., Lemmermann, N.A., ten Brinke, A., Jonjic, S., Polic, B., 2013. NKG2D induces Mcl-1 expression and mediates survival of CD8 memory T cell precursors via phosphatidylinositol 3-kinase. J. Immunol. 191, 1307–1315. Zhang, T., Lemoi, B.A., Sentman, C.L., 2005. Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood 106, 1544–1551. Zhang, T., Barber, A., Sentman, C.L., 2007. Chimeric NKG2D modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways. Can. Res. 67, 11029–11036. Zloza, A., Kohlhapp, F.J., Lyons, G.E., Schenkel, J.M., Moore, T.V., Lacek, A.T., O’Sullivan, J.A., Varanasi, V., Williams, J.W., Jagoda, M.C., Bellavance, E.C., Marzo, A.L., ´ B., Al-Harthi, L., Sperling, A.I., Guevara-Patino, ˜ Thomas, P.G., Zafirova, B., Polic, J.A., 2012. NKG2D signaling on CD8+ T cells represses T-bet and rescues CD4+ unhelped CD8 T cell memory recall but not effector responses. Nat. Med. 18, 422–428.