Influence of natural killer cells and perforin-mediated cytolysis on the development of chemically induced lung cancer in A/J mice
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- Frese-Schaper, M., Keil, A., Yagita, H. et al. Cancer Immunol Immunother (2014) 63: 571. doi:10.1007/s00262-014-1535-x
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One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4+ or CD8+ T cells, CD11b+ macrophages, Gr-1+ neutrophils and asialo GM1+ natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However, neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease.
KeywordsLung cancerNatural killer cellsPerforin-mediated cytolysisTumour necrosis factorsApoptosisImmunogenicity
Non-small cell lung cancer
Tumor necrosis factor
Tumor necrosis factor-related apoptosis-inducing ligand
TNF-like weak inducer of apoptosis
Lung cancer is the leading cause of cancer death worldwide among men and the second leading cancer site in women. In fact, 1.6 million of people were diagnosed with lung cancer in 2008 accounting for 13 % of total cases. In the same year, 1.4 million patients died from lung cancer according to 18 % of total cancer deaths . Despite improved surgery, radio- and chemotherapy, the 5-year survival remains low with approx. 16 % , suggesting that alternative treatment options are needed for this disease.
Such an alternative treatment strategy might encompass immunotherapy which has been successfully applied for other types of cancer such as prostate cancer  or melanoma . A prerequisite for the administration of immunotherapy to certain types of cancer is their immunogenicity which describes the property of cancer cells to induce a detectable reaction of the immune system. Whether lung cancer is sufficiently accessible to the immune system is an unsolved issue yet; 80 % of lung cancer is associated with smoking . Early studies demonstrated that carcinogens from tobacco smoke provoke an impaired function of the immune system. In this context, a decrease in NK cell function was found in both mice and humans [5, 6]. However, an impaired function of immune effector cells by tobacco smoke would not exclude an immunogenicity of lung tumours. Some preliminary answers to the question whether lung cancer is able to induce a sufficient reaction of the immune system were provided by vaccination studies. These studies made use of tumour antigens such as MAGE (melanoma-associated antigen)-A3 or Mucin 1 which are known to be overexpressed in non-small cell lung cancer (NSCLC) and associated with poor prognosis [7, 8]. Phase II clinical trials were conducted using these proteins for vaccination. Albeit trends for a positive clinical response to both vaccines were detectable, both studies did not reach their primary endpoint in terms of significantly prolonged survival [9, 10]. Combining the results of these clinical trials together with the demonstrated immunosuppression by tobacco smoke, these data indicate that lung cancer is not or only a weak inducer of an immune response. However, this conclusion might be amended by an encouraging clinical trial using the vaccine belagenpumaucel-L (LucanixTM) which is a mixture of allogeneic NSCLC cell lines stably transfected to secret an antisense nucleotide to TGF-β. This trial demonstrated a survival benefit of patients with NSCLC as well as an immune reaction to the vaccine .
The aim of the present work was to further shed light into the matter how the immune system is involved in the protection from lung cancer. To this end, we used a mouse model of chemically induced lung cancer. A/J mice treated with the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) developed adenomas/adenocarcinomas of the lung [12, 13] and are a well-known mouse model mainly used for chemoprevention studies. In this context, it was shown that certain substances such as isothiocyanates or methoxsalen which are believed to inhibit the mutagenic activation of NNK were able to decrease tumour growth in A/J mice [14, 15]. Also, FTI-276 an inhibitor of the enzyme farnesyltransferase which facilitates the activation of the proto-oncogene Ras demonstrated beneficial effects in the A/J mouse model . The effect of FTI-276 was attributed to mutations of the K-ras gene shown for a high percentage of lung tumours in A/J mice [17, 18]. However, only a few data exist about how the immune system contributes to the carcinogenesis in A/J mice. Splenic and peripheral NK cell number seems to be lower in A/J mice compared with C57BL/6 mice , and their cytolytic activity was shown to be lower compared to strains of mice which are not susceptible for the development of lung cancer . Moreover, administration of NNK was reported to further decrease the activity of splenic NK cells in A/J mice, an effect which was converted by the administration of non-steroidal anti-inflammatory drugs . Since non-steroidal anti-inflammatory drugs were additionally shown to inhibit tumour growth in NNK-induced A/J mice , NK cells were attributed to play a role in the carcinogenesis of this tumour model. Whether other immune cells contribute to the development of tumours in A/J mice and which mechanisms might be utilized has been investigated in the study presented here.
Materials and methods
NNK was purchased from Toronto Research Chemicals, Canada. The stock solution was prepared by solving 100 mg NNK in 200 μl DMSO. The stock solution was further diluted to a final volume of 10 ml with saline. From this solution, 200 μl (2 mg NNK) were injected to mice. For depletion of immune cells, the following antibodies were used: rat anti-CD4, clone GK 1.5 ; rat anti-CD8, clone 53-6.7 [23, 24]; rat anti-CD11b, clone 5C6 ; and rat anti-Gr-1, clone RB6-8C5 , all produced in the laboratory of Hideo Yagita. Rabbit anti-asialo GM1 purchased from Wako Pure Chemical Industries, Osaka, Japan, was used for the depletion of NK cells . Cytokines of the TNF family were depleted by the following antibodies: rat anti-TRAIL, clone N2B2 ; rat anti-TWEAK, clone MTW-1 ; and hamster anti-FasL, clone MFL4 , all produced in the laboratory of Hideo Yagita. In addition, rat anti-TNF, clone V1q  which was prepared in the laboratory of Werner Falk was used for neutralization of TNF-α. Control groups were treated with rat IgG or rabbit IgG (both from Sigma, St. Louis, MO) or hamster IgG (Jackson ImmunoResearch, Suffolk, UK).
Handling of mice
All animal experiments were approved by the Kantonale Tierversuchskommission (Kanton Bern, Switzerland). Five-week-old female A/J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in isolated ventilated cages. Treatment was applied as described below. All applications of antibodies and NNK were performed as i.p. injections. After completion of the treatment, mice were killed by an overdose of pentobarbital, and lungs were harvested. The tumour number was counted, and the size of tumours was measured using a digital calliper. The latter experiments were all executed with two investigators in which the person who measured tumour number and volume was blinded.
Generation of perforin-deficient A/J mice
Perforin-deficient (PKO) mice on a C57BL/6 background  were kindly provided by Hans Hengartner (Institute of Experimental Immunology, University of Zurich, Switzerland). These mice were backcrossed for ten generations with A/J mice. Mice with mutated perforin allele were identified by PCR analysis as described before . In brief, genomic DNA was isolated from ear biopsies using the DNeasy Blood and Tissue kit from QIAGEN (Valencia, CA, USA). For the PCR, two different primer pairs were used. 5′-TTT TTG AGA CCC TGT AGA CCC A-3′, 5′-GCA TCG CCT TCT ATC GCC TTC T-3′) which gives no product for the wild-type (wt) allele and which produces a band of 665 bp for the mutated perforin allele. 5′-CCG GTC CTG AAC TCC TGG CCA A-3′, 5′-CCC CTG CAC ACA TTA CTG GAA G-3′ yields a 300-bp fragment for wt and a 1,300-bp fragment for the mutated allele. Only the second primer pair was able to distinguish between mice harbouring a heterozygous or homozygous mutation of the perforin allele. Perforin-deficient A/J mice mice used in this study will be archived and distributed as live mice or frozen material via the European Mouse Mutant Archive (EMMA), which is part of the INFRAFRONTIER Research Infrastructure (www.infrafrontier.eu) under the strain name A.B6-Prf1tm1Sdz/Biat (EMMA ID EM:07765).
Analysis of Foxp3-positive regulatory T cells (Tregs or CD4+Foxp3+ cells) and myeloid-derived suppressor (CD11+Gr-1+) cells
Splenocytes from wt A/J mice and A/J mice deficient for perforin were freshly isolated as described before . Cells were then incubated with Fc receptor-blocking mAb (clone 2.4G2, BD Biosciences) followed by incubation with fluorochrome-labelled mAbs. For Tregs, after staining for CD4, cells were fixed and permeabilized followed by incubation with the anti-Foxp3 Ab. Staining was measured on an LSRII flow cytometer (BD Biosciences) and analysed using FlowJo software (Tree Star, Ashland, OR).The following Abs were used: anti-CD4 (clone RM4-5, Caltag), anti-Foxp3 (clone 150D, BioLegend), anti-CD11 (clone M1/70, BioLegend) and anti-Gr-1 (clone RB6-8C5, Caltag).
NK cell-sensitive Yac-1 cells (ATCC, Manassas, VA) were used as target cells. Yac-1 cells were labelled with 5-(and-6)-carboxyfluorescein diacetate succinimydyl ester (CFSE) using the 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit from Abcam (Cambridge, UK). Labelled Yac-1 cells were then added to a 96-well plate with 1 × 105 cells per well. As effector cells, freshly isolated splenocytes from wt A/J mice, AJ.PKO+/− or AJ.PKO−/− mice were added at an effector/target ratio of 20:1. The plate was centrifuged for 4 min at 30×g to allow cell contact followed by incubation for 4 h at 37 °C. Immediately before analysis, samples were incubated with 7-amino-actinomycin D (7-AAD), and death of target cells was subsequently determined by flow cytometry analysing the number of CFSE+/7-AAD+ events. The final experiments were realized in a manner that the conducting person was blinded for the groups of mice from which splenocytes were isolated.
Differences in tumour number and volume were evaluated for significance using Student’s t test or Mann–Whitney U test. A P < 0.05 was considered statistically significant.
Depletion of NK cells promotes tumour development in A/J mice
Death-inducing cytokines of the TNF family are not involved in NK cell-mediated tumour suppression in A/J mice
Tumour suppression in A/J mice depends on perforin-mediated cytotoxicity
The immunogenicity of lung cancer remains an unsolved question until now. As referred above, most of the clinical trials for lung cancer using vaccination strategies failed to achieve their primary end points. This problem might have two reasons: One could be that the antigens used for vaccination were not ideal and the other could be that lung cancer is in general not able to induce a sufficient reaction of the immune system. Our study was delineated to elucidate the second issue whether lung cancer has the ability to provoke an immune reaction or not.
In a first set of experiments, A/J mice which were induced for lung cancer with NNK were depleted of different types of immune cells. However, depletion of CD4+ T cells, CD8+ T cells, CD11b+ macrophages or Gr-1+ neutrophils did not significantly affect the tumour burden in A/J mice. These results were not expected because the deficiency of Foxp3-positive regulatory T cells was shown to reduce tumour growth in A/J mice . Tregs are a subset of CD4-positive T cells. Since we depleted in our experiments all CD4-positive cells, one might ask whether a proposed beneficial effect of losing these Tregs had been negotiated by the loss of effector CD4+ T cells. Therefore, we do not exclude from our data that in wild-type A/J mice CD4-positive cells other than Tregs contribute to the suppression of lung cancer.
Our results further demonstrated that depletion of NK cells resulted in an increased tumour number, suggesting a pivotal role of NK cells for the control of tumour growth in this lung cancer model. NK cells execute their cytotoxic effects either by direct target cell lysis or by secretion of cytokines such as interferon-γ, but without the need of antigen-specific recognition as required for cytotoxic T lymphocytes. In this context, NK cell activity was shown to be important for the development or rejection of MHC class-I-deficient lymphomas , and depletion of NK cells was also found to promote tumour growth of fibrosarcomas . Furthermore, there is evidence that lung cancer development depends on the function of NK cells. In a recent publication, Kreisel and colleagues demonstrated that depletion of NK cells promoted urethane-induced lung tumour growth in a mouse strain which is normally not susceptible to lung cancer . Moreover, NK cells were shown to have lower activity in A/J mice , which are prone to develop lung cancer induced by certain chemicals such as the nitrosamine NNK. NNK itself was demonstrated to further reduce NK cell activity in A/J mice , an effect which has been observed in human smokers as well . Despite the assumption of lower NK cell activity in NNK-induced A/J mice, our data suggest that the remaining NK cell activity was sufficient to reduce lung tumour formation. However, once the lung tumours are established, another situation might arise comprising a direct inhibition of NK cell activity by lung cancer cells. Thus, it has been shown that lung tumour cells from malignant pleural effusions inhibit NK cell activity , and it was further demonstrated that NK cells isolated from lung tumours exhibit decreased cytotoxic activity .
The mechanisms how NK cells induce direct target cell lysis might vary; both the engagement of cytokines of the TNF family as well as the perforin-mediated granule exocytosis pathways are known . Many studies were published showing a significant contribution of TNF family cytokines to the protection from different types of cancer. While the presence of TRAIL and FasL was demonstrated to have anti-cancer attributes [41–43], the genetic ablation or antibody-mediated depletion of TNF-α inhibited the development of skin cancer [44, 45]. Similar to TNF-α, TWEAK seems to act in a pro-cancerogenic manner . However, in our experimental setup, neither the neutralization of TRAIL, FasL, TWEAK or TNF-α alone nor in combination had an influence on NNK-induced lung tumour development. This was somewhat surprising especially for TRAIL since our previous laboratory works provided promising results when treating lung cancer cells with this cytokine [47, 48]. As an alternative pathway, we found in vivo and in vitro evidence that the perforin-mediated pathway contributes to NK cell-mediated tumour suppression in A/J mice, as perforin-deficient A/J mice exhibited increased tumour development and reduced cytolytic NK cell activity. Moreover, the number of other immunosuppressive cells such as Tregs was not different in perforin-deficient mice compared to their wild-type counterpart what might speak against a significant contribution. However, the effect of PKO on tumour growth was less pronounced than the effect of NK cell depletion. Therefore, the involvement of other factors such as IFN-γ or lymphotoxin-α which were shown before to be involved in NK cell-mediated cytotoxicity cannot be excluded [49, 50].
Collectively, our data suggest a significant contribution of NK cells to the suppression of lung cancer. This finding might have important consequences since NK cell activity can be stimulated using the glycolipid α-galactosylceramide (α-GalCer), which activates invariant NKT cells and dendritic cells to produce IFN-γ and IL-12, respectively. Subsequent activation of NK cells by IFN-γ and IL-12 is again accompanied by profound production of IFN-γ . The effects of α-GalCer on NKT and NK cells were further explored in clinical trials. While systemic administration of α-GalCer induced a prolonged depletion of NKT cells from peripheral blood , the ex vivo activation of respective NK cells using α-GalCer is another promising approach. A phase I/II study including patients with advanced or recurrent NSCLC has been published recently with encouraging results. A prolonged survival was observed in patients who showed increased IFN-γ production in peripheral blood mononuclear cells stimulated with α-GalCer . However, further studies are needed especially in an adjuvant setting of radically resected T1 and T2 NSCLC in order to see whether stimulation of NK cells lowers the risk of local or metastatic recurrence.
We thank Beatrice Zumkehr for technical assistance. We are also thankful to Hans Hengartner for providing a breeding pair of PKO mice on a C57BL/6 background. This work was supported by the Bernische Krebsliga and by the Stiftung für Klinisch-Experimentelle Krebsforschung Bern, both grants to Steffen Frese.
Conflict of interest