Natural Killer Cell Lytic Activity Research Articles

NK Cell Regulation of CD4 T Cell-Mediated Graft-versus-Host Disease

CD3-negative NK cells are granular lymphocytes capable of producing inflammatory cytokines and killing malignant, infected, or stressed cells. We have recently observed a new role for NK cells in the control of the proliferation of CD4 T cells under persistent antigenic stimulation. Monoclonal anti-male CD4 T cells transferred into Rag2-/- male recipients did not expand or were rapidly eliminated. Remarkably, T cells transferred into NK cell-deficient Rag2-/- Il-2Rgammac-/- male hosts expanded extensively and mediated tissue lesions usually observed in chronic graft-versus-host disease (GVHD). T cell failure to proliferate and to induce chronic GVHD was the result of NK cell activity, because depletion of the recipient's NK1.1+ cells by Ab treatment induced T cell expansion and chronic GVHD. T cells under chronic Ag stimulation upregulated ligands of the activating receptor NKG2D, and regulatory activity of NK cells was inhibited by the injection of Abs directed to NKG2D. On the contrary, blocking NKG2A inhibitory receptors did not increase NK cell regulatory activity. Finally, we show that NK regulation of T cell expansion did not involve perforin-mediated lytic activity of NK cells, but depended on T cell surface expression of a functional Fas molecule. These results highlight the potential role played by NK cells in controlling the Ag-specific CD4+ T cells responsible for chronic GVHD.

In vitro preclinical study results for a phase I adoptive immunotherapy trial using cetuximab with allogeneic NK cells in KRAS oncogene-mutated metastatic colorectal cancer patients.

e14057 Background: Prognosis for metastatic colorectal-cancer (mCRC) patients is unfavorable. Futhermore mCRC patients with tumors harboring a mutation in the KRAS gene do not derive benefit from the administration of cetuximab. We hypothesized that KRAS mutated CRC cells may be susceptible to cetuximab-induced ADCC mediated by healthy donor NK cells. Methods: Purified allogeneic NK cells or preincubated with IL-2 or IL-15 were used as effectors. Seven different CRC cell lines (DLD1, HCT-116, HT-29, LOVO, SW620, SW48, SW480) labeled with 51Cr were used as target cells alone or preincubated with cetuximab for ADCC assay. Results: All but one (SW620) cell lines express EGFR and MHC molecules; 4/7 harbor KRAS mutation (DLD 1, HCT-116, SW620, SW480). All cell lines were susceptible to unstimulated NK cells lysis (mean 20% ± 4.57, at ratio 25:1; mean 15% ± 3.65, at ratio 12:1) and lytic activity is strongly enhanced by IL-2 or IL-15 (mean 48% ± 5.8, at ratio 25:1; mean 38% ± 4.3, at ratio 12:1) (p < 0.05). No difference in lytic activity was observed between IL-2 vs. IL-15 and thawed vs. fresh NK cells. The SW48, a KRAS wilde-type cell line was killed by NK cells from 38% of lysis without cetuximab to 50% with cetuximab (E/T ratio of 50:1, p < 0.05). Similarly the HCT-116, a KRAS mutated cell line was killed by NK cells from 20% without cetuximab to 36% with cetuximab (E/T ratio of 50:1, p < 0.05). When IL-2 or IL-15 activated NK cells were used as effectors, both targets lysis increased from 50% without cetuximab to 71% with cetuximab (E/T ratio of 100:1, p < 0.05). When SW620 (EGFR expression 0%) was used as target cell line, pre-coating with cetuximab did not enhanced unstimulated or pre-activated NK lysis capacity. Conclusions: allogeneic NK cells are cytotoxic versus all tested cell lines. Moreover lytic activity of thawed and fresh NK cells is similar. In ADCC assay NK cell lytic activity is significantly enhanced by cetuximab; surprisingly cetuximab mediated ADCC activity is independent from CRC cell lines KRAS oncogene mutational status and require EGFR membrane expression. Moreover lytic activity of IL-2 and IL-15 activated NK cells is increased by cetuximab. No significant financial relationships to disclose.

Abstract B11: Hyperthermia and antitumor immunity

Abstract Background: A phase III randomised EORTC trial combining systemic chemotherapy and regional hyperthermia (RHT) for patients (n=341) with locally-advanced high-risk soft tissue sarcoma (STS) showed that – compared to chemotherapy alone – the addition of RHT yields a statistically significant improvement in tumor response, with doubling of disease free and local progression free survival (ASCO 2007). A phase II study in chemo-pretreated patients with STS receiving 2nd line ICE chemotherapy (ifosfamide + carboplatin + etoposide) showed clinical activity with no further toxicity if combined with RHT (ASCO 2009). The biological rationale of this approach is based on the direct cytotoxicity of heat and its chemosensitizing effect on tumor cells. Cells respond to heat by producing increasing amounts of molecular chaperones (heat shock proteins, HSPs). Some HSPs, in particular HSP70, are heat-inducible and are released by necrosis, where outside the cells they gain potent immunostimulatory functions. We provided strong evidence that heat up-regulated expression of HSP70 correlates with enhanced susceptibility to NK cell lysis of tumor cells. Besides recognizing of HSP70 on tumors as a target structure, it has been shown that NK cells can be activated ex vivo by the HSP70-derived 14-mer peptide (TKD), enhancing NK immune function. A phase I clinical study showed that reinfusion of autologous NK cells activated ex vivo by TKD in patients with advanced disease is well tolerated, feasible and initiates cytolytic activity of NK cells. In summary, this is a strong rationale that hyperthermia might have a positive impact on the immune system within the context of the heat shock response. We are implementing a prospective randomised study that investigates the impact of autologous TKD-activated NK cells adoptively transferred in patients with high-risk STS responding to chemotherapy and RHT. In a preliminary study we investigated the number and lytic activity of NK cells in patients with high-risk STS. Patients and methods: Patients at primary diagnosis were scheduled to receive an anthracyclin and ifosfamide-based first-line chemotherapy and RHT; patients with progressive disease were scheduled to receive a 2nd line regimen (ICE) and RHT. Informed consent was obtained from all patients. Blood samples taken before the start of therapy were investigated for changes in lymphocyte subpopulations and for their lytic activity cells before and after therapy. Results: Between April 2007 and December 2008 7 1st-line and 14 2nd-line patients were enrolled. 1. A progressive reduction in absolute NK cell counts but no relevant changes in total lymphocyte counts were observed over the applied cycles in second-line patients. 2. Whereas NK mediated lytic activity against K562 is detectable in healthy donors, the spontaneous NK cell activity in all but one second-line patients was completely abrogated at the start of the therapy and was not restored over 4 cycles of ICE+RHT. 3. First-line patients also showed a significant decrease in NK cell activity compared to healthy donors 4. In vitro stimulation of patients' NK cells with IL-2/TKD induced a highly significant increase in NK cell activity against K562 cells in first- and second-line patients, indicating that NK cells preserve their ability to be activated in vitro. Most interestingly, this ability persisted over the applied cycles of thermochemotherapy. Conclusions: STS patients have suppression of NK cell activity but this lytic activity can be repristinated in vivo after stimulation with TKD/IL-2. Translational studies are performed to further elucidate the potential role of hyperthermia in promoting an antitumor immune response. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B11

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells

Tumor resistance to lysis by resting natural killer (NK) cells may be overcome by priming of NK cells with cytokines or by binding of NK activating receptors to ligands expressed on target cells. In this study, major histocompatibility complex class I (MHC-I)-negative LNCaP and MHC-I-positive DU145 cells were infected with genetically modified influenza A virus lacking the non-structural gene 1 (NS1 IAV). The cells were used to investigate the influence of NS1 IAV infection on NK cell lysis of tumor cells as well as to prime NK cells for lysis of LNCaP and DU145 cells. While LNCaP cells infected with DeltaNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% +/- 1.47 vs. 52% +/- 0.74), both mock-infected and DeltaNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with DeltaNS1 IAV-infected LNCaP/DU145 cells effectively lysed resistant DU145 and sensitive LNCaP cells to a greater extent than NK cells primed with mock-infected LNCaP/DU145 or non-primed NK cells. Also, NK cell priming with DeltaNS1 IAV-infected tumor cells enhanced extracellular signal-regulated kinase phosphorylation and increased granule release in NK cells. The increased granule release was specifically mediated by NKp46, which eventually potentiated NK cells primed with DeltaNS1 IAV-infected tumor cells to overcome the inhibitory effects posed by MHC-I expression on DU145 cells. These findings show that in addition to direct lytic activity of NK cells, DeltaNS1 IAV may influence anti-tumoral responses by priming NK cells.

Prevention of Mammary Carcinogenesis in MMTV-neuMice by Cruciferous Vegetable Constituent Benzyl Isothiocyanate

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of human breast cancer cells in culture. The present study provides in vivo evidence for efficacy of BITC for prevention of mammary cancer in MMTV-neu mice. Administration of BITC at 1 and 3 mmol/kg diet for 25 weeks markedly suppressed the incidence and/or burden of mammary hyperplasia and carcinoma in female MMTV-neu mice without causing weight loss or affecting neu protein level. For example, cumulative incidence of hyperplasia/carcinoma was significantly lower in mice fed BITC-supplemented diets compared with control mice (P = 0.01 by Fisher's test). The BITC-mediated prevention of mammary carcinogenesis correlated with suppression of cell proliferation and increased apoptosis. The average number of Ki-67-positive cells in the carcinoma lesions of 3 mmol BITC group was lower by approximately 21% (P < 0.05) compared with tumors from control mice. Apoptotic bodies in the mammary tumor were higher by about 2- to 2.5-fold in the 1 and 3 mmol BITC treatment groups (P < 0.05) compared with control group. The BITC administration also resulted in overexpression of E-cadherin and infiltration of CD3(+) T-cells in the tumor. Although BITC treatment increased cytotoxicity of natural killer (NK) cells in vitro, dietary feeding of BITC failed to augment NK cell lytic activity in an ex vivo assay. The present study demonstrating efficacy of BITC against mammary cancer in an animal model provides impetus to determine its activity in a clinical setting.

In Vitro Preclinical Study Results for a Phase I Adoptive Immunotherapy Trial Using Cetuximab in Association with Allogeneic NK Cell Infusion in KRAS Oncogene Mutated Metastatic Colorectal Cancer Patients.

Abstract 5119 PurposePrognosis for metastatic colorectal-cancer (mCRC) patients is unfavorable, median survival from diagnosis varying between sixteen and twenty-four months. Numerous clinical trials have demonstrated a clinical advantage for patients treated with a combination of chemotherapy and cetuximab. However recent retrospective evidence from several randomized studies, has established that mCRC patients with tumors harboring a mutation in the KRAS gene do not derive benefit from the administration of cetuximab. We hypothesized that KRAS mutated CRC cells may be susceptible to cetuximab-induced ADCC mediated by healthy donor NK cells. Experimental Design7 different CRC cell lines (DLD 1, HCT-116, HT-29, LOVO, SW620, SW48, SW480) were analyzed for EGFR membrane expression levels, tested for KRAS mutational status and MHC class I-II expression. CD56+CD3- NK cells were purified from volunteer healthy donors peripheral blood mononuclear cells, by a two-steps CD3+ cells negative selection followed by CD56+ cells positive selection by MACS cell separation columns (Miltenyi biotec). Purified NK cells were incubated overnight with medium alone or stimulated with IL-2 (100U/ml) or IL-15 (20U/ml). CRC cell lines, were labeled with 51Cr overnight, and used as target cells for NK assay or incubated at 4°C for 30 minutes with Cetuximab 100 ug/ml for ADCC assay. Cytotoxic activity was evaluated in a 4-hour cytotoxicity assay, at an effector/target (E/T) ratio ranging between 100:1 and 2:1. Resultsall but one (SW620) CRC cell lines express EGFR (range 40-90%) and MHC class I-II molecules; 4/7 harbor KRAS mutation (DLD 1, HCT-116, SW620, SW480). All CRC cell lines were susceptible to unstimulated allogeneic NK cells lysis (mean 20% ± 4,57, at ratio 25:1; mean 15% ± 3,65, at ratio 12:1) and lytic activity is significantly enhanced (p<0.05) by either IL-2 or IL-15 pre-activation (mean 48% ± 5,8, at ratio 25:1; mean 38% ± 4,3, at ratio 12:1, respectively). No difference in lytic activity was observed between IL-2 vs IL-15 pre-activation and between thawed vs fresh NK cells. SW48, KRAS wilde-type (EGFR expression 74%) and HCT-116 KRAS mutated (EGFR expression 61%) cell lines were chosen as targets for ADCC assay. When unstimulated NK cells were used as effectors, SW48 lysis increased from 38% without cetuximab pre-coating to 50% with cetuximab pre-coating (E/T ratio of 50:1, p<0.05). Similarly, HCT-116 lysis increased from 20% without cetuximab pre-coating to 36% with cetuximab pre-coating (E/T ratio of 50:1, p<0.05). When IL-2 or IL-15 pre-activated NK cells were used as effectors, both targets lysis increased from 50% without cetuximab pre-coating to 71% with cetuximab pre-coating (E/T ratio of 100:1, p<0.05). Pre-coating with cetuximab did not enhanced unstimulated or pre-activated NK lysis against SW620 (EGFR expression 0%). Conclusionsallogeneic NK cells from different healthy donors are able to lyse all tested CRC lines (4 KRAS mutated). IL-2 and IL-15 activation significantly enhances NK cytotoxicity; moreover lytic activity of thawed and fresh NK cells is similar allowing the use of both populations in clinical trials. In ADCC assay NK cell lytic activity is significantly enhanced by cetuximab; surprisingly cetuximab mediated ADCC activity is independent from CRC cell lines KRAS oncogene mutational status and require EGFR membrane expression. Pre-coating with cetuximab also increase lytic activity of IL-2 and IL-15 activated NK cells. Further experiments are in progress to evaluate the susceptibility to the lysis of primary tumor cells derived from KRAS mutated and wild type CRC patients, in NK and ADCC assays in order to confirm data obtained against established CRC cell lines and to correlate cetuximab-mediated ADCC with EGFR membrane expression. These findings support a phase I study with allogeneic NK cells infusions in association with cetuximab and IL-2 in mCRC patients harboring a mutation in the KRAS gene. DisclosuresNo relevant conflicts of interest to declare.

Phenoxodiol, an anticancer isoflavene, induces immunomodulatory effects in vitro and in vivo

Phenoxodiol (PXD) is a synthetic analogue of the plant isoflavone genistein with improved anticancer efficacy. Various properties and mechanisms of action have been attributed to the drug, the most important being its ability to sensitize resistant tumour cells to chemotherapy, which led to its fast track FDA approval for phase II/III clinical trials. In this study, we examined the effects of PXD on human peripheral blood mononuclear cells (PBMC) and its potential role in regulating immune responses. We show that PXD, at concentrations ≥1 μg/ml (4 μM), inhibited proliferation and reduced the viability of healthy donor-derived PBMC. In contrast, lower PXD concentrations (0.05–0.5 μg/ml) augmented, upon 3-day incubation, PBMC cytotoxicity. Experiments with purified CD56+ lymphocytes revealed that PXD enhanced the lytic function of natural killer (NK) cells by directly stimulating this lymphocytic subpopulation. Furthermore, in an in vivo colon cancer model, Balb/C mice administered low-dose PXD, exhibited significantly reduced tumour growth rates and prolonged survival (in 40% of the animals). Ex vivo results showed that PXD stimulated both NK and tumour-specific cell lytic activity. We conclude that PXD, when administered at low concentrations, can act as an immunomodulator, enhancing impaired immune responses, often seen in cancer-bearing individuals.

Sulforaphane Inhibits Prostate Carcinogenesis and Pulmonary Metastasis in TRAMP Mice in Association with Increased Cytotoxicity of Natural Killer Cells

The present study shows that oral gavage of 6 mumol d,l-sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived L isomer, thrice per week beginning at 6 weeks of age, significantly inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice without causing any side effects. The incidence of the prostatic intraepithelial neoplasia and well-differentiated (WD) carcinoma were approximately 23% to 28% lower (P < 0.05 compared with control by Mann-Whitney test) in the dorsolateral prostate (DLP) of SFN-treated mice compared with controls, which was not due to the suppression of T-antigen expression. The area occupied by the WD carcinoma was also approximately 44% lower in the DLP of SFN-treated mice relative to that of control mice (P = 0.0011 by Mann Whitney test). Strikingly, the SFN-treated mice exhibited approximately 50% and 63% decrease, respectively, in pulmonary metastasis incidence and multiplicity compared with control mice (P < 0.05 by t test). The DLP from SFN-treated mice showed decreased cellular proliferation and increased apoptosis when compared with that from control mice. Additionally, SFN administration enhanced cytotoxicity of cocultures of natural killer (NK) cells and dendritic cells (DC) against TRAMP-C1 target cells, which correlated with infiltration of T cells in the neoplastic lesions and increased levels of interleukin-12 production by the DC. In conclusion, the results of the present study indicate that SFN administration inhibits prostate cancer progression and pulmonary metastasis in TRAMP mice by reducing cell proliferation and augmenting NK cell lytic activity.

Small doses of morphine can enhance NK cell cytotoxicity in pigs

The effect of small and moderate doses of morphine (MF) on NK cell lytic activity (cytotoxicity, NKCC) ( 51Cr release test) and the number of circulating large granular lymphocytes (LGL) was evaluated in i.v. catheterized Pietrain crossbred pigs. Simultaneously, plasma cortisol (COR) (RIA method) was measured. Blood samples were collected 15, 60, 120, 180, and 240 min after i.v. injections of 0.5, 1.0 and 5.0 mg/kg of MF alone or MF pretreated with naloxone (NX, 1.0 mg/kg, i.v., 15 min before MF). It was found that MF induced dose- and time-dependent changes of NKCC. MF in a dose of 0.5 mg/kg evoked 4-fold increase in NKCC (in comparison to saline) without changes in the number of LGL/NK cells. Higher MF doses (1.0, 5.0 mg/kg) induced an early increase (up to 300Δ% and 29Δ%, respectively) followed by a decrease in cytotoxicity (to − 76Δ% after 5.0 mg/kg), and in LGL number (− 36Δ% after 5.0 mg/kg). These effects were concomitant with a marked rise in plasma COR (up to 234Δ% after 0.5 mg/kg and 567Δ% after 5.0 mg/kg of MF). NX pretreatment blocked all the changes in cytotoxicity but not in the LGL cell number and COR concentrations. The results indicate that MF, besides having well known immunosuppressive effects, can also enhance NKCC through the opioid receptors-dependent manner. The enhancement of cytotoxicity appears as a purely functional change independent of the recirculation of NK cells which occurs despite the high plasma concentrations of COR.

The Role of Interleukin-15 on Natural Killer Cell Homeostasis and Activity in Patients with Acute Myeloid Leukemia

Interleukin-15 (IL-15) has been demonstrated to play a critical role in the regulation of natural killer (NK) cells. IL-15 induces the differentiation of NK cells from hematopoietic progenitors, stimulates the expansion of peripheral NK cells, and supports their survival. We investigated the role of IL-15 as a homeostatic regulator of NK cells in 29 patients diagnosed with acute myeloid leukemia (AML) and the potential role of IL-15 in enhancing the anti-tumor activity of NK cells in AML patients. The percentage of circulating NK cells was lower (p<0.0001) in the AML patients (6%± 0.7, range 1–17%) compared to the NK cells of healthy donors (12%± 1, range 9–17 %). At diagnosis the mean level of IL-15 in patient plasma was 1.9 pg/ml (range 0.03–8.9) and increased (p <0.02) to 5.2 pg/ml (range 0.06–13.4) after the completion of induction chemotherapy, when the NK levels had been reduced to zero cells/microliter. The mean level of IL-15 subsequently decreased to pre-treatment levels in the AML patients who achieved complete remission (mean 1.6 pg/ml, range 0.4–2.3). To assess effects of IL-15 on the NK cytotoxicity, we sorted NK cells from PBMC obtained from AML patients prior to treatment (at diagnosis) and cultured them in the presence of IL-15. Following IL-15 stimulation, a significant increase in NK-cell cytotoxicity against K562 targets and the patients' autologous leukemic blasts was observed (p<0.05) as was up-regulation in expression of the activating natural cytotoxicity receptors, NKp30 and NKp46 and the C-type lectin receptors NKG2D and NKG2C (p<0.02–0.001). Addition of blocking antibodies to the activating receptors reduced NK-cell cytotoxicity. We determined that IL-15, a homeostatic NK-cell cytokine, increases after severe depletion of NK cells following intensive chemotherapy and this leads to increased NK-cell lytic activity in AML patients. These data suggest that modulation of IL-15 levels in AML could be therapeutically beneficial as IL-15 enhances NK-cell recovery following intensive chemotherapy and increases NK-cell anti-tumor activity.

Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by photosensitivity, a high incidence of cancer in sun-exposed portions of the skin and a reduced capacity to repair the u.v.-induced DNA damage. One of the XP mutations (XP-D) has also been identified in patients affected by trichothiodystrophy (TTD), a rare autosomal recessive disease characterized by brittle hair, mental and physical retardation, peculiar face and ichthyosis. However, in these patients there is no evidence of increased skin tumour incidence. Since an impairment of cell-mediated immunity has been proposed as a co-factor in the cancer proneness of XP patients, we investigated the involvement of immune defect(s) in five XP patients, five TTD patients, their parents, and 24 TTD relatives. We evaluated the phenotype of circulating lymphocytes, natural killer (NK) cell lytic activity, target cell binding of NK cells at single cell level and the effect of interferons (IFN) alpha and beta on NK cell activity. The relative proportion of CD3+ and CD4+ circulating lymphocytes was reduced in XP but not in TTD patients. NK cell lytic activity was decreased in XP patients and their mothers, but their fathers showed normal lytic activity. NK activity varied among TTD families: four out of five patients and their relatives presented low NK cell activity, and one family was normal. In TTD family members, NK activity increased after incubation with IFN-alpha or IFN-beta, but never reached normal values. In contrast, in XP patients and their mothers, the defect was almost completely corrected after in vitro incubation with IFN-alpha or IFN-beta. Our study indicates impaired NK lytic activity in the majority of TTD and XP patients and that this defect is present also in members of their families. In addition, XP patients present a low number of circulating T cells. These multiple abnormalities, together with DNA repair defects, could be related to the increased cancer risk in XP patients.

Increased NK cell lytic activity in chronic HCV infection

Increased NK cell lytic activity in chronic HCV infection

Anti‐tumor effect by dendritic cells stimulated with IL‐2 in OT‐1 model

Background:Recently, IKDCs expressing both natural killer (NK) and dendritic cell (DC) characteristics have been described: these NK‐like DCs “convert” from cells with NK cell lytic activity and NK receptor expression, into cells acquiring MHC class II and the requisite molecules for APC function. In an attempt to stimulate IKDC‐like activity in bone‐marrow‐derived DCs (BMDCs), we stimulated the BMDCs with IL‐2. These “DCs‐IL‐2” cells possessed an impressive anti‐tumor effect in mice when injected s.c, mixed with M05 tumor cells.Methods:1) Vaccination with DCs‐IL‐2+MO5 in OT‐1 model; 2) Rechallenge of tumor‐free mice; 3) Flow and image analysis.Results:1) The DCs‐IL‐2‐treatment demonstrated an impressive anti‐tumor effect in that no tumor growth was seen in four of the five mice that received DCs‐IL‐2 + MO5 tumor cells on the right flank and M05 cells alone on the left flank; 2) Three out of the four tumor‐free mice did not grow tumors when re‐challenged with MO5 tumor cells. One mouse developed tumors, but much delayed compared to control mice; 3) Flow data demonstrated that 10–20% of the DCs‐IL‐2 acquired A‐NK phenotypic markers, while maintaining their DC phenotypes.Conclusion:The DCs‐IL‐2 have a promising potential to improve cancer immunotherapy and we will continue to study the phenotype, lineage commitment, and in vivo relevance of these cells.

Natural Killer cell function during foot-and-mouth disease virus infection (46.19)

Abstract Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. Immunological knowledge to assess more rapid acting vaccines against FMDV is presently limited. We examined the reactivity of swine and cattle NK cells following infection for their capability to express intracellular perforin, to kill a human tumor cell line target in vitro, and to secret IFN gamma. The cytotoxicity of NK cells from non-infected animals against the K562 cells is low with baseline levels at 5–15% in swine and 15–20% in cattle. Stimulation with rhIL-2 or rhIL-12 plus rhIL-15, increased the lytic activity against K562 cells. Infection with FMDV inhibited swine NK cell lytic activity but did not significantly increase IFN gamma secretion during the acute infection. Perforin expression increased but this did not correlate with the killing capability of the swine NK cells. Infection of cattle with FMDV initially activated the NK cells to increase target cell lysis. NK cell IFN gamma secretion and perforin expression were slightly elevated upon infection and coincided with the lytic activity in cattle. These results are a further indication of immune evasion by FMDV by inhibiting or limiting NK cell function. The potential to manipulate the innate immune response to block this evasion is discussed in the context of designing rapid acting vaccines for foot-and-mouth disease.

Interleukin-21 Enhances NK Cell Activation in Response to Antibody-Coated Targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.

A half-marathon and a marathon run induce oxidative DNA damage, reduce antioxidant capacity to protect DNA against damage and modify immune function in hobby runners

This study investigated whether a 21.1 km (half-marathon) or a 42.195 km (marathon) run modulates DNA damage, antioxidant capacity in lymphocytes and plasma, and the immune system in healthy hobby runners. Ten and 12 volunteers who completed the Baden-Marathon race in Karlsruhe with a running distance of 21.1 km and 41.195 km, respectively, were assessed 10 days before and immediately after the finish. There was no increase in the levels of endogenous DNA strand breaks immediately after half-marathon or marathon races. A statistically significant increase in the levels of oxidative DNA damage in lymphocytes was found using endonuclease III but not formamidopyrimidine glycolase (Fpg). The resistance of DNA to oxidative damage induced by hydrogen peroxide in isolated lymphocytes was significantly decreased after both races. The levels of plasma antioxidants such as α-tocopherol, β-carotene and lycopene were close to, or higher than, those considered optimal for reducing the risk of cardiovascular diseases and there were no significant changes after the races in antioxidant capacity of LDL (lag-time test) or plasma in ORAC, TEAC or paraoxonase assays. The number and percentage of granulocytes and monocytes able to generate oxidative burst were significantly increased after both races, but the lytic activity of NK cells was significantly increased at the end of the half-marathon; no effect was observed in the marathon runners. Thus, oxidative DNA damage in lymphocytes, decreased the antioxidant capacity to protect lymphocytes against DNA strand breaks and increased the formation of reactive species by phagocytes in well-nourished hobby runners indicating moderate oxidative damage during such high-intensity exercise.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Prevention of Relapse after Chemotherapy in a Chronic Intracellular Infection: Mechanisms in Experimental Visceral Leishmaniasis

In visceral leishmaniasis, chemotherapy probably seldom eradicates all parasites in tissue macrophages; nevertheless, most T cell-intact patients show long-lasting clinical cure after treatment despite residual intracellular infection. To characterize prevention of posttreatment relapse, amphotericin B was used to kill approximately 90-95% of Leishmania donovani in livers of mice deficient in mechanisms of acquired antileishmanial resistance. Recrudescence subsequently developed 1) in animals deficient in both CD4 and CD8 T cells as well as CD40L-mediated T cell costimulation, but not in a) CD4 or CD8 cells alone, b) NK cell lytic activity, or c) ICAM-1-recruited monocytes; and 2) in mice deficient in IFN-gamma, but not in the IFN-gamma-inducing cytokines, a) IL-12, b) IL-12 and IL-23, or c) IL-18. Posttreatment recrudescence also did not develop in animals deficient in macrophage phagocyte NADPH oxidase (phox) or inducible NO synthase (iNOS) alone or, surprisingly, in those deficient in both phox and iNOS. Therefore, regulation of the intracellular replication of residual Leishmania donovani that escape chemotherapy evolves to a host mechanism distinguishable from initial acquired resistance at the T cell, cytokine, and macrophage levels. Posttreatment, either CD8 or CD4 cells can direct the response, IL-12 is not required, and iNOS and phox, the activated macrophage's primary IFN-gamma-inducible leishmanicidal pathways, both become dispensable.

CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma

To investigate the effect of CpG-containing oligodeoxynucleotides (CpG ODN) alone or in combination with the chemotherapeutic agent 5-fluorouracil (5-FU) on tumor growth and whether CpG ODN can reverse the immunosuppression caused by the chemotherapy with 5-FU in murine hepatoma model. Hepatoma model was established by subcutaneous inoculation with hepatoma-22 (H(22)) cells into the right flank of BALB/c mice. Mice with tumor were treated by peritumoral injection of CpG ODN alone or in combination with subcutaneous injection of 5-FU. Tumor size was quantified regularly. Serum levels of IL-12 and IFN-gamma in mice were assayed by enzyme-linked immunosorbent assay (ELISA). The lytic capacity of splenic NK cells was tested by lactate dehydrogenase release assay. Peritumoral injection of CpG ODN alone or in combination with subcutaneous injection of 5-FU, and the treatment with 5-FU alone all led to significant inhibition of hepatoma growth. The mean tumor volumes fell by 46.66% in mice injected with CpG ODN, 68.34% in the 5-FU treated mice, and 70.23% in mice treated with the combination of CpG ODN and 5-FU than in controls. There was no significant difference in tumor size between 5-FU-treated mice and mice treated with the combination of 5-FU and CpG ODN (P>0.05). The serum levels of IL-12 and IFN-gamma of mice treated with CpG ODN alone (IL-12: 464.50+/-24.37 pg/mL; IFN-gamma: 134.20+/-25.76 pg/mL) or with the co-administration of CpG ODN and 5-FU (IL-12: 335.83+/-28.74 pg/mL; IFN-gamma: 111.00+/-5.33 pg/mL) were significantly higher than that of controls (IL-12: 237.50+/-45.31 pg/mL; IFN-gamma: 56.75+/-8.22 pg/mL). The production of IL-12 and IFN-gamma was suppressed moderately in 5-FU-treated mice (IL-12: 166.67+/-53.22 pg/mL; 53.33+/-16.98 pg/mL) compared to control mice (P>0.05), whereas the combination of CpG ODN and 5-FU significantly increased the serum levels of IL-12 and IFN-gamma compared to 5-FU alone (P<0.05). The NK cell killing activity in CpG ODN-treated mice (44.04+/-1.38%) or the mice treated with CpG ODN combined with 5-FU (30.67+/-1.28%) was significantly potentiated compared to controls (19.22+/-0.95%, P<0.05). The co-administration of CpG ODN and 5-FU also significantly enhanced the lytic activity of NK cells when compared with the treatment with 5-FU alone (12.03+/-1.42%, P<0.05). The present data suggests that CpG ODN used as single therapeutic agent triggers anti-tumor immune response to inhibit the growth of implanted hepatoma and reverses the immunosuppression caused by the chemotherapy with 5-FU.

Consumption of purple sweet potato leaves modulates human immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production

To study the immunological effects of physiological doses of purple sweet potato leaves (PSPL). The randomized crossover study (two periods, each lasting for 2 wk) involved 16 healthy non-smoking adults of normal weight. The 6-wk study consisted of a run-in (wk 1) PSPL diet (daily consumption of 200 g PSPL) or a control diet (low polyphenols, with the amount of carotenoids adjusted to the same level as that of PSPL) (wk 2-3), washout diet (wk 4), and switched diet (wk 5-6). Fasting blood was collected weekly in the morning. T-lymphocyte function was assessed via the proliferation and secretion of immunoreactive cytokines. Salivary IgA secretion and the specific cytotoxic activities of cytotoxic T lymphocytes and natural killer (NK) cells were determined. The plasma beta-carotene level increased with time in both groups, while the plasma polyphenol level decreased in the control group, and no significant difference was detected between the two groups. Although plasma polyphenol levels did not significantly increase in the PSPL group at the end of the study, they were significantly elevated in urine. PSPL consumption produced a significant increase in proliferation responsiveness of peripheral blood mononuclear cells (PBMC) and their secretion of immunoreactive IL-2 and IL-4. As well, lytic activity in NK cells was elevated in a time-dependent fashion. Salivary IgA secretion significantly decreased in control group after 2 wk, and returned to baseline following dietary switch to PSPL. Consumption of PSPL modulates various immune functions including increased proliferation responsiveness of PBMC, secretion of cytokines IL-2 and IL-4, and the lytic activity of NK cells. The responsible determinants of PSPL remain to be elucidated, as does the biological significance of the present observations.