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Enhancement of natural killer (NK) cell cytotoxicity by feverrange thermal stress is dependent on NKG2D function and is associated with plasma membrane NKG2D clustering and increased expression of MICA on target cells Julie R. Ostberg,1,2 Baris E. Dayanc,1 Min Yuan, Ezogelin Oflazoglu, and Elizabeth A. Repasky3 Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York, USA Abstract: Circulating NK cells normally experience temperature gradients as they move about the body, but the onset of inflammation can expose them and their targets to febrile temperatures for several hours. We found that exposure of human peripheral blood NK cells and target cells to feverrange temperatures significantly enhances lysis of Colo205 target cells. A similar effect was not observed when NK cell lines or IL-2-activated peripheral blood NK cells were used as effectors, indicating that thermal sensitivity of effectors is maturation or activation state-dependent. Use of blocking antibodies revealed that this effect is also dependent on the function of the activating receptor NKG2D and its ligand MHC class I-related chain A (MICA). On NK cells, it was observed that thermal exposure does not affect the total level of NKG2D surface expression, but does result in its distinct clustering, identical to that which occurs following IL-2-induced activation. On tumor target cells, a similar, mild temperature elevation results in transcriptional up-regulation of MICA in a manner that correlates with increased sensitivity to cytolysis. Overall, these data reveal that NK cells possess thermally responsive regulatory elements, which facilitate their ability to capitalize on reciprocal, stress-induced changes simultaneously occurring on target cells during inflammation and fever. J. Leukoc. Biol. 82: 1322–1331; 2007. Key Words: inflammation 䡠 innate immunity 䡠 IL-2 䡠 febrile temperatures 䡠 hyperthermia activating versus inhibitory signals through the ligation of specific receptors. Activation is also associated with “clustering” of the activating receptors in lipid raft regions of the plasma membrane [3]. Specifically, experimental evidence shows that the early and what is sometimes called the “nonspecific phase” of NK cell activation are regulated by the proinflammatory milieu of the microenvironment [4, 5]. Certain cytokines, including IL-2, IL-7, IL-12, IL-15, and Type I IFNs [4, 6 –9], are known to activate NK cells rapidly, and TNF-␣ has been found specifically to be involved in the recruitment of NK cells to inflammatory sites [10, 11]. It is at this point, after a nonspecific activation has resulted from cytokine inflammatory mediators, that NK cell-mediated killing must also be triggered by the dominance of activation receptor complexes over inhibitory receptor complexes [12–14]. It is important that some of the same proinflammatory cytokines (e.g., TNF-␣, IFN-␣/) involved in the early events leading to activation of NK cells are also pyrogenic, causing an increase in body temperature or fever through several overlapping physiological, behavioral, and neurological changes (reviewed in refs. [15, 16]); this increase in body temperature is considered one of the cardinal features of inflammation and takes place in the same general time-frame in which NK cells are initiating their activation and functional activity, as both events are triggered by the same proinflammatory signals. However, despite the temporal linkage between cytokinedriven NK cell activation and the pyrogenicity of these same cytokines, a separate thermal component in the regulation of NK cell activation has not been fully characterized. Is it possible that at least some of the physiological effects of local inflammatory cytokines on NK cell function are mediated indirectly through a thermal shift? If so, how do cells “recognize” thermal signals in the activation pathway leading to cytotoxicity, and how is specificity for appropriate targets maintained INTRODUCTION NK cells provide essential, innate host protection against viral infections and tumor formation. Not only do these cells kill target cells directly, but they also have the ability to manipulate downstream, long-term, adaptive immune responses [1, 2]. Recent work has revealed remarkable complexity in the regulation of NK cell activation and cytotoxic potential. Indeed, NK cell activation and killing only occur after exposure to inflammatory cytokines and after achieving the correct balance of 1322 Journal of Leukocyte Biology Volume 82, November 2007 1 These authors contributed equally to this work. Current address: Division of Cancer Immunotherapeutics and Tumor Immunology, City of Hope National Medical Center, Beckman Research Institute, Duarte, CA, USA. 3 Correspondence: Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. E-mail: [email protected] Received November 24, 2006; revised July 6, 2007; accepted July 25, 2007. doi: 10.1189/jlb.1106699 2 0741-5400/07/0082-1322 © Society for Leukocyte Biology when millions of cells well beyond the initial inflammatory site are affected by an increase in body temperature? When others examined the effects of thermal stress on human NK cell cytotoxic activity, use of temperatures significantly above fever-range (i.e., ⬎42°C or 107°F) was found to generally inhibit NK cytotoxicity [17–20], and more mild elevations in temperature (i.e., more similar to fever-range) have been associated with enhanced NK cytotoxic activity [21–23]. Indeed, earlier studies by our group [24] using various murine tumor models suggested that the ability of a mild, fever-range, whole body hyperthermia (39.5– 40°C for 6 – 8 h) to inhibit tumor growth in vivo was a result of the action of NK cells based on use of function-blocking antibodies. However, in a physiological situation, elevated temperatures would affect both effectors and targets. To date, the requirement for heating the NK effector cells and/or targets has never been examined, nor has the mechanisms by which temperature affects NK cell activation been defined. Thus, to begin to address these issues, we have examined the effects of temperature on effector cells and target cells using freshly isolated human peripheral blood NK cells or NK cell lines and human Colo205 tumor cells. We report here that cytotoxic activity of human peripheral blood NK cells (but not long-term cultured cell lines, nor cells activated by IL-2) is optimally enhanced when NK effectors and tumor target cells are exposed to mild hyperthermic conditions. We also describe the mechanistic linkage of this effect to the involvement of the activating receptor NKG2D on NK cells and its MHC class I-related chain A (MICA) ligand on tumor target cells. These data strongly suggest that peripheral blood NK cells are responsive to the evolutionarily conserved temperature gradients associated with fever/inflammation and may have evolved to take advantage of simultaneous, reciprocal, stress-induced alterations on the surface of target cells exposed to the same temperature gradient in the body. MATERIALS AND METHODS Cell lines and human NK cell isolation Human NK cells were isolated from healthy donor peripheral blood by density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich Inc., St. Louis, MO, USA) and purified by negative magnetic separation using a NK cell isolation kit (Miltenyi Biotech, Auburn, CA, USA), according to the manufacturer’s protocol. Cell purity was found to be ⬎95%. The human colon adenocarcinoma cell lines Colo205, HCT116, and HT29 [American Type Culture Collection (ATCC), Manassas, VA, USA] were propagated in RPMI-1640 medium with 2 mM L-glutamine and 10% FBS. The human NKL cell line, a generous gift from Dr. Jerome Ritz (Dana Farber Cancer Institute, Boston, MA, USA), was propagated in RPMI 1640 with L-glutamine, sodium pyruvate, 15% FBS, and 100 IU/mL human recombinant IL-2 (Sigma-Aldrich Inc.). The human NK cell line NK92MI, a malignant, non-Hodgkin’s lymphoma, transfected with human IL-2 cDNA (ATCC), was propagated in MEM-␣ modified with ribonucleosides, deoxyribonucleosides, and 2 mM L-glutamine, supplemented with 0.2 mM inositol, 0.1 mM 2-ME, 0.02 M folic acid, 12.5% horse serum, and 12.5% FBS. Viability of all cells in culture, even IL-2-starved NKL cells, was ⬎90% for each experiment. Cytotoxicity assays Cytotoxicity assays were performed using the CytoTox 96 nonradioactive cytotoxicity assay (Promega Corp, Madison WI, USA), according to the manufacturer’s protocol. In some cytotoxicity assays, cells were incubated with 1 g/mL anti-NKG2D or 5 g/mL anti-MICA/B F(ab)2, or blocking antibodies, or 6 g/mL isotype control, IgG (R&D Systems, Minneapolis, MN, USA) for 15 min before mixing with target cells. In other cytotoxicity assays, cells were incubated with 5 mM methyl- cyclodextrin (MCD) or DMSO vehicle control during the last 30 min of a 6-h culture at 37°C or 39.5°C and then washed to remove drug prior to coculture of cells in a 6-h cytotoxicity assay. The maximum and spontaneous release was determined by incubating the target cells and effector cells with 0.5% Triton X-100 or medium alone, respectively. In all experiments, spontaneous release of effectors and targets was ⬍15% of total release. Percent cytotoxicity was determined using the following equation: 100 ⫻ (experimental release–spontaneous release)/(maximum release–spontaneous release). mAbs and flow cytometry For flow cytometric staining, 106 cells were washed with PBS before being incubated with anti-NKG2D (R&D Systems), anti-MICA (Immatics Biotechnologies, Tubingen, Germany), PE-conjugated anti-HLA ABC (BD Biosciences PharMingen, San Diego CA, USA), or isotype control antibodies for 30 min on ice. Cells incubated with nonconjugated primary antibodies were then washed and stained with PE-conjugated goat anti-mouse secondary antibody (BD Biosciences PharMingen) for 20 min on ice. Cells were then fixed with freshly prepared 2% paraformaldehyde in PBS and analyzed within 12 h with a FACScan (BD Biosciences, San Jose, CA, USA) and WinMDI 2.8 software. Immunofluorescent staining for NKG2D localization Freshly purified human NK cells were centrifuged at 300 g for 5 min, washed with cold PBS, and fixed with 2% paraformaldehyde in PBS for 15 min at 4°C prior to incubation with 10 g/mL mouse anti-human NKG2D mAb (BD Biosciences PharMingen) for 1 h at 4°C. Cells were then washed with cold PBS, incubated with 10 g/mL FITC-conjugated goat anti-mouse polyclonal antibody (BD Biosciences PharMingen) for 1 h at 4°C, washed with cold PBS, and resuspended in Vectashield mounting media with 4⬘,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame CA, USA) for analysis by confocal microscopy (Leica Microsystems AG, Wetzlar, Germany). RNA preparation, RT-PCR, and real-time quantitative PCR (qPCR) procedure RNA was extracted from 107 cells using the RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) and quantified by measuring absorbance at 260 and 280 nm using the GeneQuant RNA/DNA calculator (Pharmacia/Pfizer, New York, NY, USA). Extracted, total RNA (1 g) was mixed with 0.5 g oligo-dT 12–18 primers (Molecular Probes Inc., Invitrogen, Carlsbad, CA, USA), 10 mM deoxy-(d)NTP mix, and water, incubated at 65°C for 10 min, chilled on ice, and centrifuged briefly. The mixture was combined with 5⫻ first-strand buffer, DTT, RNase inhibitor, and SuperScript II RT (Molecular Probes Inc., Invitrogen), incubated at 42°C for 50 min and at 70°C for 15 min. The resulting cDNA was then combined with PCR buffer (10 mmol/L Tris-HCl, 50 mmol/L KCL), 1.5 mmol/L MgCl2, 2.5 mmol/L each dNTP and 2.5U Taq polymerase (Molecular Probes Inc., Invitrogen), as well as 10 pmol each MICA sense and antisense primers. Amplification included 30 cycles of denaturation (95°C for 1 min), annealing (56°C for 1 min), and extension (90 s at 72°C). GAPDH message was amplified and used as an internal control. PCR products were analyzed with 1.5% agarose gel electrophoresis, ethidium bromide staining, and the BioDoc-It transilluminator system (UVP, Upland, CA, USA). Relative quantification was performed using the comparative threshold (CT) method. For qPCR experiments, SYBR Green I dye (Molecular Probes Inc., Invitrogen) was used according to the manufacturer’s protocols with MICA reverse (5⬘GCAGGGAATTGAATCCCAGCT-3⬘) and forward (5⬘-ACACCCAGCAGTGGGGGGAT-3⬘) or GAPDH reverse (5⬘-TCCACCACCCTGTTGCTGTA-3⬘) and forward (5⬘-ACCACAGTCCATGCCATCAC-3⬘) primers and 96-well qRTPCR plates (Axygen Scientific Inc. Union City, CA, USA). For real-time quantitative detection, an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used, and fold change in relative MICA signal was determined using the 2–⌬⌬Ct formula against the GAPDH signal. Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress 1323 ELISA for soluble MICA (sMICA) Culture supernatants were harvested at the indicated times and analyzed for sMICA levels using a human MICA DuoSet ELISA development kit (R&D Systems) as per the manufacturer’s instructions. RESULTS Enhancement of NK cell cytotoxic activity by mild thermal stress Here, we report on the existence of a thermally responsive component in the regulation of NK cell function, one that appears to significantly enhance the cytotoxic activity of peripheral blood-derived, human NK cells against tumor targets. In a series of experiments, freshly isolated human NK cells from the peripheral blood of normal donors were cocultured with human colon tumor target cells (Colo205) at 33°C, which reflects skin temperature at the body’s extremities, the normothermic temperature of 37°C, or physiologically relevant, febrile temperatures of 38°C (lower febrile range) or 39.5°C (upper febrile range) during a 6-h cytotoxicity assay. Each of these was then also compared with coculture of cells at 42°C for 1 h, which reflects a classical heat shock condition, followed by incubation at 37°C for the remaining 5 h of a 6-h assay. Although some experiments indicated an enhancement of cytotoxicity at 38°C using this time-frame (data not shown), a consistently significant alteration in cytotoxic activity compared with the 37°C cultures was observed when the cells were cultured at 39.5°C for 6 h (Fig. 1A). If these cells were heated at 39.5°C for 6 h and then allowed to recover for 24 h at 37°C Fig. 1. Temperatures influence the cytotoxic activity of peripheral blood-derived human NK cells. Freshly purified human NK cells were incubated for 6 h with Colo205 human colon adenocarcinoma cells (A–C) or autologous PBMCs (D) at 33°C (e), 37°C (䡬), 38°C (f), or 39.5°C (Œ) or at 42°C for the 1st hour before continuing the culture at 37°C (●). (A–C) Data acquired with NK cells from three different human donors. Target cell killing was assayed with a lactate dehydrogenase (LDH) release assay. All data are expressed as average percent specific cytotoxicity ⫾ SE of triplicate wells and are representative of three independent experiments. *, P ⬍ 0.05, when comparing 39.5°C with 37°C control values at each E:T ratio using an unpaired Student’s t-test. 1324 Journal of Leukocyte Biology Volume 82, November 2007 Fig. 2. Activation with IL-2 correlates with inability of mild thermal stress to enhance cytotoxic activity of human NK cell lines and purified human NK cells. Human NK cell lines NKL (A) and NK92MI (B) or peripheral blood-derived human NK cells activated with IL-2 (100 IU/ml) for 2 days (C) were incubated for 6 h with Colo205 human colon adenocarcinoma cells at 37°C (䡬) or 39.5°C (Œ). NKL cells were also starved of IL-2 for 2 h prior to coincubation with Colo205 targets (D). Target cell killing was assayed with a LDH release assay. All data are expressed as average percent specific cytotoxicity ⫾ SE of triplicate wells and are representative of three independent experiments. *, P ⬍ 0.05, when comparing control and heated values at each E:T ratio using an unpaired Student’s t-test. before coculture in a 6-h cytotoxicity assay, a complete loss of the enhanced cytotoxic activity was observed (data not shown). Examination of the time-frame required at 39.5°C to get an enhanced cytotoxic effect revealed that 4 h of coculture was not always enough to see statistically significant enhancement of cytotoxic potential, and 8 h of coculture did not appear to be any better than 6 h (data not shown). Indeed, despite interpersonal variation among donors, statistically significant increases in cytotoxic efficiency were consistently observed with NK cells collected from the peripheral blood of various healthy donors and incubated with targets for 6 h at 39.5°C (Fig. 1, B and C). This thermal enhancement of NK cell cytotoxic activity was highly specific and dependent on the presence of an appropriate activating NK target. Thermal enhancement of cytotoxicity was not observed when coheated, autologous PBMCs were used as targets (Fig. 1D), indicating that there is no nonspecific induction of NK cytolytic activity by heat; i.e., heat does not appear to release lytic molecules nonspecifically, which should kill all cells in the environment. Instead, target cells bearing appropriate ligands must be present. Moreover, when human NK cell lines such as NKL (Fig. 2A) and NK92MI (Fig. 2B) were used as effectors with tumor targets, they displayed no enhancement of cytotoxicity with mild thermal stress. Conversely, if the NKL cells were starved of IL-2 immediately prior to coculture with the Colo205 targets, thermal enhancement of cytotoxic activity was then observed (Fig. 2D). As these cell lines might be considered more highly activated than peripheral blood-derived NK cells, in part through their constitutive culture with IL-2 and as evidenced by their higher percent http://www.jleukbio.org cytotoxicity values when cultured at 37°C (compare Fig. 1, A–C, with Fig. 2, A or B), we also examined the effects of mild thermal stress on human peripheral blood NK cells, which had been cultured with IL-2. These “preactivated” NK cells (from a human donor, which had displayed thermal enhancement of cytotoxicity previously in the absence of IL-2) now revealed an inability to be thermally stimulated (Fig. 2C). Thus, overall, it appears that the cytotoxic activity of freshly derived human peripheral blood-derived NK cells, but not NK cell lines or IL-2-activated human NK cells, is sensitive to this mild, physiologically relevant elevation in temperature. However, as shown here, even when using freshly isolated, peripheral blood-derived NK cells, thermal enhancement is not seen, unless there is an appropriate target (i.e., tumor cells but not autologous PBMC). Thermal enhancement of NK cytotoxicity requires thermal stimulation of NK cells and tumor targets To determine whether NK cells, tumor cells, or both were responsive to these temperature changes, we next preincubated the NK effectors and tumor cells separately at 37°C or 39.5°C for 6 h before placing them together in a 6-h cytotoxicity assay at 37°C. Although incubation of just the target cells at elevated temperatures appeared to enhance cytotoxicity slightly more than incubation of just the NK cells at elevated temperatures (Fig. 3A), it was observed that the most consistent and significant enhancement of cytotoxicity occurred when NK cells and Colo205 tumor cells underwent preheat treatment. This suggests that both the effectors and targets can be affected by changes in their thermal microenvironment in a manner that increases the overall cytotoxic response of NK cells against tumor cell targets. Dependence of NK cytotoxicity on NKG2D and its ligand MICA NKG2D is one of the major activating receptors on NK cells, which mediate cytotoxic activity by recognizing defined ligands, often overexpressed on stressed or transformed cells [25]. To test the hypothesis that the function of this receptor was necessary for thermal enhancement of cytotoxicity, we used a blocking antibody against NKG2D (Fig. 3B). Addition of this function-blocking antibody not only significantly inhibited the cytotoxicity observed when cells were incubated at normo- thermic temperatures (as expected), but thermally enhanced cytotoxicity was also lost completely. This suggests that the cellular pathway by which NK cells respond to thermal signals in terms of enhanced cytotoxicity ultimately still depends on NKG2D receptor function. These data also support the conclusion drawn from data shown earlier that there is no nonspecific release of lytic molecules by heat without engagement of appropriate targets. Similar to heat shock protein genes, the 5⬘ end flanking regions of the NKG2D ligands MICA and MICB, in particular, are known to harbor putative heat shock response elements, which permit their up-regulation upon heat shock at 42°C [26]. Thus, this potential regulation of MICA by heat shock led us to investigate the dependence of the thermally induced cytotoxicity of Colo205 cells on this ligand. Using a blocking antibody against MICA, there was only a partial (although significant) loss of cytotoxic activity observed with the heated cells (data not shown). However, this result could be misleading because of the potential for whole IgG molecules against MICA to mediate FcR-associated, antibody-dependent cell-mediated cytotoxicity by the NK cells. Thus, blocking experiments were also carried out with F(ab)2 molecules against MICA (Fig. 3C). This resulted in complete inhibition of the thermal effect on cytotoxicity and suggests that MICA is the predominant NKG2D ligand by which mild thermal stress mediates the enhancement of NK cell cytotoxic activity. Regulation of NKG2D membrane localization but not total surface expression by mild thermal stress As blocking antibodies against NKG2D were found to inhibit basically all thermally induced, NK-mediated, cytotoxic activity, we suspected that the cellular mechanism by which thermal signals were mediated ultimately depended on this receptor. Thus, in our search for an underlying mechanism for the thermal enhancement of NK cytotoxicity, we began by examining the effects of mild thermal stress on this activating receptor. As NK cells have been found to display increased expression of NKG2D when they are treated in vitro with the stimulatory agent IL-15 [27], we first examined the effects of mild thermal stress on NKG2D expression levels on NK cells. Flow cytometric analysis revealed no effect of culture at 39.5°C Fig. 3. Enhanced cytotoxicity requires treatment of both human NK cells and Colo205 targets with elevated temperatures and involves NKG2D and its ligand MICA. (A) Cells were precultured separately at 37°C or 39.5°C for 6 h prior to coincubation at 37°C during the cytotoxicity assay. NK cells and target cells precultured at 37°C (䡬); NK cells cultured at 39.5°C and targets at 37°C (䉬); target cells cultured at 39.5°C and NK cells at 37°C (f); NK cells and target cells precultured at 39.5°C (Œ). Data are expressed as average percent specific cytotoxicity ⫾ SE of triplicate wells. A representative of three different experiments, each performed using NK cells from three different donors, is shown. *, P ⬍ 0.05, using Student’s t-test to compare NK cells and target cells precultured at 37°C (䡬) versus NK cells and target cells precultured at 39.5°C (Œ) at each E:T ratio. (B and C) Cells were incubated for 6 h at 37°C (open bars) or 39.5°C (solid bars) prior to addition of isotype control IgG or anti-NKG2D-blocking antibody (B) or anti-MICA-blocking F(ab)2 fragments (C) at 37°C and performance of cytotoxicity assays. Data are expressed as average percent-specific cytotoxicity ⫾ SE of triplicate wells at a 10:1 E:T ratio. Representative data of four experiments are shown. †, P ⬍ 0.05, comparing heated to nonheated cultures under each condition using a Student’s t-test. *, P ⬍ 0.05, comparing IgG to blocking mAb or F(ab)2 conditions within the heated or nonheated cultures using a Student’s t-test. Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress 1325 Fig. 4. NKG2D surface localization, but not overall expression levels, are altered by mild thermal stress. (A) Freshly purified human NK cells were cultured for 6 h at 37°C (solid line) or 39.5°C (dashed line) and then stained with anti-NKG2D or isotype control antibody (filled histogram). (B) Freshly purified human NK cells were cultured for 6 h at 37°C (C), 39.5°C (HT), or 37°C with 10 g/mL cross-linking antiNKG2D antibody (Ab) for 15 min before being fixed, stained for NKG2D, and visualized by confocal microscropy. These were compared with human NK cells, which had been activated with 100 IU/mL IL-2 for 2 days (IL2), as well as NKL cells, which are propagated in 100 IU/mL IL-2 (NKL). Representative cells with nonclustered NKG2D after culture at 37°C, with clustered NKG2D after culture at 39.5°C, or with clustered NKG2D after culture with crosslinking antibodies are depicted. (C) Upon visualizing 50 or more cells in each group, average percentages of cells with surface clustering of NKG2D were determined in n ⫽ 3 experiments ⫾ SE. (D) Percentages of cells with clustered surface NKG2D when heated cells were allowed to recover at 37°C for 12 (⫹12 h) and 24 (⫹24 h) h were also determined as described for C. *, P ⬍ 0.05, when compared with control values using a Student’s t-test. on overall NKG2D surface expression on human NK cells (Fig. 4A). However, previous studies in our laboratory have shown that exposure of cells to mild thermal stress can alter the physical positioning of several different membrane-associated molecules in a manner associated with heightened activation potential. These include alterations in the organization of the spectrin-based cytoskeleton and several PKC isoforms as well as the formation of uropods [28, 29]. As redistribution or clustering of activating receptors at the site of target recognition is known to occur when NK cells form conjugates with target cells is known to occur during IL-2-enhanced NK cell cytotoxicity [3], we next examined the ability of mild thermal stress to affect NKG2D localization on the NK cell surface (Fig. 4, B and C). It is notable that a significant increase in cells with aggregates of clustered NKG2D on the cell surface was observed after NK cells were exposed to mild thermal stimulation compared with those which were incubated at normothermic temperatures. Although addition of anti-NKG2D cross-linking antibody resulted in a larger number of cells with clustered NKG2D, the significant increase in the number of cells with an identical pattern of clustered NKG2D at 39.5°C as that seen with IL-2 treatment strongly supports the hypothesis that membrane localization of NKG2D is highly sensitive to mild temperature shifts in a manner that enhances cytotoxic ability. Furthermore, a reversal of the thermally induced NKG2D clustering was seen within 24 h after the heated, purified human NK cells were returned to 37°C (Fig. 4D). This loss of the clustered phenotype correlated directly with a reversal of the enhanced cytotoxic activity observed when these cells, which had been allowed to recover for 24 h at 37°C, were then cultured with tumor cell targets (data not shown). The same analysis of NKG2D positioning on IL-2-treated peripheral bloodderived human NK cells and the NK cell line NKL revealed that even at normothermic temperatures, there was a higher percent of cells with NKG2D clustering, similar to that seen in NKG2D 1326 Journal of Leukocyte Biology Volume 82, November 2007 cross-linked cells; yet there was no further increase seen after mild thermal stress (Fig. 4C and data not shown). As clustering of NKG2D at the cell surface is known to be related to the association of this receptor with lipid raft domains in the plasma membrane [3], we next examined the ability of MCD to affect thermally induced cytotoxicity. MCD is known to cause cholesterol to effuse from the membrane and thereby disrupt lipid raft organization, and this reagent has been used by others to determine the dependence of an event upon the integrity of lipid rafts [30]. Thus, MCD was added to the last 30 min of the 6-h culture of cells at 39.5°C versus 37°C, and then the drug was washed away before targets and effectors were cocultured at 37°C for the cytotoxicity assay. Cell survival was not affected, but surface, heatinduced protein clustering was eliminated by this MCD treatment (data not shown). Further, we found that the thermal enhancement of cytotoxicity was totally eliminated by MCD treatment (Fig. 5). These data suggest that normal lipid raft structure and organization are required for the thermally induced effects on NK-mediated cytotoxic activity, and that the ability of the activating receptor NKG2D to cluster at the membrane surface is at least part of the mechanism by which NK cells respond to fever-like thermal stress. Regulation of MICA expression by mild thermal stress Based on the anti-MICA F(ab)2-blocking studies described above, as well as the potential regulation of MICA by heat shock, we also investigated the ability of a physiologically relevant, mild thermal stress of 39.5°C to affect MICA expression in Colo205 cells. RT-PCR analysis revealed significantly enhanced MICA mRNA expression in the tumor cells within 3 h of incubation at 39.5°C (Fig. 6). This enhancement of the MICA mRNA levels appeared to plateau between 4 h and 8 h of culture at this fever-range temperature (data not shown). http://www.jleukbio.org Fig. 5. Thermal enhancement of cytotoxicity is lost with MCD treatment. Colo205 cells were incubated for 6 h at 37°C (open bars) or 39.5°C (solid bars) with a final concentration of 5 mM. MCD was added during the last 30 min prior to washing the cells and performance of cytotoxicity assays. Data are expressed as average percent-specific cytotoxicity ⫾ SE of triplicate wells at a 20:1 E:T ratio. Representative data of four experiments are shown. †, P ⬍ 0.05, comparing heated to nonheated cultures under each condition using a Student’s t-test; *, P ⬍ 0.05, comparing controls to MCD treatment within the heated or nonheated cultures using a Student’s t-test. Flow cytometric analysis then revealed enhanced surface MICA expression on Colo205 cells cultured at 39.5°C for 6 h compared with those cultured at 37°C (Fig. 7A). Thermally enhanced surface expression of this NKG2D ligand was also found with the colon tumor line HCT116 (Fig. 7B) but not HT29 colon tumor cells, possibly as these cells had higher constitutive MICA expression with which to begin (Fig. 7C). Fig. 7. Elevated temperatures can enhance MICA but not MHC class I surface expression. Human colon tumor cell lines Colo205 (A), HCT116 (B). and HT29 (C), and human PBMC (D) or freshly purified human NK cells (E) were cultured for 6 h at 37°C (gray line) or 39.5°C (thick, black line) and then stained with anti-MICA (left) or anti-MHC class I (HLA-ABC, right) or isotype control antibody (thin, black line). Fig. 6. Elevated temperatures enhance MICA mRNA levels in Colo205 cells. (A) Total RNA was extracted from Colo205 cells after incubation at 37°C for 6 h or cultured at 39.5°C for 1, 3, and 6 h, and MICA and GAPDH message was detected with RT-PCR. (B) MICA message is quantified with real-time qRT-PCR. Bars represent fold induction in MICA message over 37°C control ⫾ SE of triplicate samples. All data are representative of three independent experiments. *, P ⬍ 0.05, when compared with 37°C controls using a Student’s t-test. Importantly that these MICA expression patterns correlated with the thermal sensitivity of these cell lines to NK-mediated killing, as the NK cytotoxicity of HCT116 was enhanced significantly by mild thermal stress [and was blocked by antiMICA F(ab)2 molecules], but that of HT29 was not (data not shown). Enhanced MICA surface expression was not observed with total human PBMCs under the same conditions (Fig. 7D), as may have been expected by the cytotoxicity results described for Figure 1D above. Detectable MICA expression was also not induced on purified human NK cells (Fig. 7E). Overall, MHC class I expression on the tumor cells, PBMC, or NK cells was also not affected by 6 h culture at 39.5°C (Fig. 7), sug- Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress 1327 Fig. 8. sMICA is not induced by mild thermal stress. Using human colon tumor cell line HCT116 (5⫻104/mL) as a positive control, culture supernatants from Colo205 cells (5⫻104/mL), freshly purified human NK cells (106/mL), or a coculture of NK and Colo205 cells were collected after 6 h at 37°C (C) or 39.5°C (HT) or after heated cells were allowed to recover for 6 (⫹6 h) or 12 (⫹12 h) h at 37°C. Data are expressed as average concentration of sMICA ⫾ SE of two to three different experiments. sMICA was not detected in all but the HCT116 supernatants; *, supernatants not tested. gesting that mild thermal stress has no obvious effect on levels of these molecules, which are known to act as inhibitory ligands for NK cytotoxicity. As soluble MIC (sMIC) proteins are known to be released by some human tumors in a manner, which appears to help them escape immune recognition [31, 32], we also examined the ability of mild thermal stress to affect levels of sMICA in the supernatants of our cultures (Fig. 8). It is interesting that mild thermal stress did not induce detectable levels of sMICA when NK cells or Colo205 cells were cultured alone, nor when they were cultured together. HCT116 cells did produce sMICA, as reported previously [33], but levels did not appear to be enhanced significantly after 6 h of culture at 39.5°C. Together, these data suggest that mild thermal stress preferentially up-regulates the surface expression of the NKG2D ligand MICA on certain colon tumor cells in a transcriptionally regulated manner. The ability of mild thermal stress to up-regulate the expression of MICA thus also appears to play an important role in determining the sensitivity of the target cell to NK-mediated cytotoxicity. DISCUSSION Circulating human immune effector cells normally briefly experience mild temperature gradients as they move through cooler regions of the skin and airway mucosal surfaces (29 – 33°C) to deeper, warmer sites maintained at core temperature (⬃37°C). The onset of fever following infection would result in a steeper thermal gradient experienced by immune effector cells (and their potential targets), as core temperature in humans typically increases by 1–3°C [34]. The studies reported here reveal the potential of a physiologically relevant thermal shift to enhance the lytic ability of NK cells against similarly heat-stressed target cells. The fact that this enhancement occurs in the febrile range and not at higher “heat shock” temperatures strongly suggests an evolutionarily conserved, physiologically relevant response to inflammatory conditions (including a universally recognized thermal component), which 1328 Journal of Leukocyte Biology Volume 82, November 2007 occur following exposure to antigens. This ability of mild thermal stress to regulate freshly derived blood NK cells but not NK cell lines, which are constitutively cultured with the activating cytokine IL-2, nor freshly derived NK cells treated with IL-2, has revealed for the first time a selective thermal sensitivity in NK cells, that is apparently dependent on the activation status of these effector cells. Furthermore, we have observed that a fever-range, mild thermal stress can enhance NK cell cytotoxic activity through membrane-associated effects on both NK cell effectors and human colon tumor targets; and we have identified the activating receptor NKG2D and its ligand MICA as primary targets in the thermal regulation of human NK cytotoxicity. These studies extend previous work by our group [35–39], suggesting 1) the potential of thermal shifts to act as a “danger signal” similar to the effects of other known danger signals [40] and 2) the ability of whole body hyperthermia to inhibit tumor growth in vivo using murine tumor models, an effect which appears to be dependent, at least in part, on the increased infiltration and function of NK cells [24]. Unlike previously identified types of danger signals, which are often molecules released from dying cells and thus, may act predominantly in a local manner (reviewed in ref. [41]), body temperature can affect a large number of effector cells at the same time, including those that are not near the inflammatory milieu or have not come into contact with any form of soluble mediator or antigen- or ligand-expressing target. Thus, there is potential for thermal stress to lower rapidly and systemically, the activation threshold of distant immune cells well in advance of their encounter with antigen or other soluble mediators of danger. However, if this notion is correct, and regional or systemic increase in temperature has the potential of affecting millions of NK cells at the same time, there must be mechanisms in place to prevent nonspecific killing of self or inappropriate target cells. We observed here several clues indicating the existence of such a regulatory mechanism in control of thermal signaling. We found that thermally enhanced, NK cell-mediated cytotoxicity is highly specific: no killing of autologous PBMC targets occurred upon heating, revealing the absence of heat-induced release of lytic substances in the absence of appropriate activation signals. Indeed, heated NK cells must still be exposed to an appropriate target and experience normal activation signals (coming from that target) for mild thermal stress to have an effect. Antibodies blocking NKG2D eliminated killing of targets completely, including any enhancement by heat. Further, significant up-regulation of MICA also helped to enhance and guide the thermally enhanced lysis of tumor targets, thus minimizing the potential for autoimmune activity. Previous in vitro studies have revealed that activation of NK cells is associated with clustering of receptors [3]. Although activation-induced clustering is traditionally thought to occur only at the contact site with a target, we propose that a morphologically similar, thermally induced receptor clustering all over the cell surface, as observed here, could still be at least part of the underlying basis by which thermal signals increase the lytic potential of NK cells. Although much work remains to identify the biophysical process by which membrane-associated molecules rearrange in response to temperature shifts, our data about the lack of detectible surface or sMICA on NK cells http://www.jleukbio.org indicate that thermally induced NKG2D clustering is not a result of sMICA or surface-expressed MICA on these freshly purified NK cells. Furthermore, the importance of thermally induced clustering of activating receptors is also supported by our observations of an activation state-dependent thermal sensitivity. There is a complete lack of fever-range thermal sensitivity/regulation in long-term NK cell lines, which are cultured in IL-2 and in purified human peripheral blood NK cells that have been preactivated with IL-2. However, long-term, cultured NK cell lines (which are known to express functional NKG2D molecules [42, 43]) and IL-2-activated NK cells were revealed here to constitutively display a higher percent of cells with similarly clustered NKG2D receptors. Thus, it is possible that in this circumstance, receptor clustering cannot be enhanced further with mild thermal stress, which may help to explain their thermal insensitivity. That enhanced cytotoxic activity of freshly purified NK cells is lost when cells are recultured at 37°C, or the cells are incubated with MCD, which disrupts lipid rafts, further supports the hypothesis that thermally induced NKG2D clustering within lipid rafts is associated directly with thermal enhancement of NK cell cytotoxicity. Up-regulation of the NKG2D ligand MICA has been reported previously to occur upon more severe stresses such as heat shock (⬎42°C) [26], bacterial or viral infection [42, 44, 45], DNA damage [46], oxidative stress [47], and treatment with retinoic acid [48] or the histone deacetylase inhibitor sodium valproate [49]. The data presented here add mild thermal stress, easily achievable under physiological conditions, to this list of conditions which can up-regulate MICA. Furthermore, although there is fever-range enhancement of surface MICA, there was no observable change in MHC class I expression or sMICA, both of which could inhibit NK cytotoxic activity, suggesting that NK cells can capitalize on this thermally induced shift in the balance of signals, resulting in increased lytic ability. Although association of higher MICA expression with higher NK-mediated cytotoxicity is not surprising, given previous data about the effects of various stressors on MICA expression [26, 42, 44 – 49], our data examining the effects of heating target cells alone (Fig. 3) suggest that such up-regulation of MICA by mild thermal stress is not sufficient to induce statistically significant enhancement of NK-mediated cytotoxicity. However, the fact that febrile temperatures are sufficient to increase MICA expression suggests the intriguing possibility that NK cells have learned to take advantage of this stress-induced effect on target cells by a reciprocal response: clustering of NKG2D receptors, which may strengthen NK cell interactions with the MICA ligand on target cells. Indeed, Figure 3A suggests that NK cells and target cells must each respond to the change in their thermal microenvironment. This is supported by the observation that although the NK cells may be induced to alter surface NKG2D localization with mild thermal stress, a lack of thermally induced enhancement of MICA/ligand expression on the target cell (as seen with HT29 cells) results in an absence of a statistically significant or discernable increase in overall cytotoxicity. Examining the effects of mild thermal stress on a broader range of tumor cell types, and determining whether fever-range temperatures enhance MICA surface expression on tumor cells and NKG2D organization on NK cells in vivo will be critical future studies. Whether there is also a thermal effect on the expression or function of inhibitory receptor molecules (i.e., killer inhibitory receptors) on NK cells and their corresponding specific ligands on target cells also needs to be examined further. The enhancement of NK cytotoxic activity at the mild hyperthermic temperature ranges shown here contrasts with previous studies, which used temperatures significantly above fever range (⬎42°C or 107°F), where NK cytotoxicity was inhibited significantly [17–20]. Indeed, our data using Colo205 cells (in Fig. 1) also show that coincubation at 42°C did not enhance cytotoxicity. This might indicate that heat shock conditions inhibit NK cell function, despite the known ability of heat shock to up-regulate MICA on tumor cells. However, our data support previous reports that indicate enhancement of human NK cytotoxic activity at lower, more physiologically relevant temperatures [21–23]. Our data also extend these observations significantly by indicating that the enhanced cytotoxicity requires heating of the target cells and is not a result of increased secretion of lytic molecules, thus providing mechanistic evidence regarding the role of NKG2D and its ligand MICA in the thermal enhancement of NK-mediated cytotoxicity. The remarkable evolutionary conservation of the febrile response [50] suggests that there is a beneficial and specific role for temperature shifts in the inflammatory process (reviewed in ref. [51]. Multiple studies suggest that application of a mild hyperthermia can enhance several endpoints of the immune system selectively in a manner which can help control infections [52–54] and the growth of tumors in murine models [24, 55]. Overall, we believe the data presented here further support the hypothesis [39] that elevations in the thermal microenvironment might serve as a danger signal, directly or indirectly (via effects on targets themselves) alerting NK cells to cellular insult and facilitating their more rapid (yet specific) lytic potential through receptor clustering and perhaps additional downstream signaling events. A systemic elevation in temperature would have the potential of alerting and/or increasing the immune-activating potential of cells over a widespread region of the body not in immediate contact with other danger-signal molecules in a local inflammatory situation. These data may also help to identify anti-tumor activity of heat-exposed NK cells as one of the factors providing significantly improved tumor control, as revealed in several recent Phase III trials using hyperthermia [56]. Although further study is required to dissect the precise mechanisms by which NK cells and other immune effector cells are able to perceive and respond to an altered physical parameter of their microenvironment (i.e., temperature), the observations described here could help in the development of new treatments, in which heat is strategically delivered in combination with immunotherapy to enhance NK cell-mediated cytotoxicity. ACKNOWLEDGMENTS This work was supported by National Institutes of Health P01 CA94045, R01 CA71599, and R21 CA098852 and the Ro- Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress 1329 swell Park Cancer Institute core grant CA16056. B. E. D. was supported by the Komen Foundation DISS0402487 and a Fulbright predoctoral grant. The authors thank Bonnie Hylander and Sarah Hejaily for their review of the manuscript and Jeanne Prendergast, Diane Thompson, and Christopher Gregorie for their expert laboratory assistance. REFERENCES 1. Kelly, J. M., Darcy, P. 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