brain tumours are intracranial neoplasms located in the brain and meninges ( excluding haematopoietic malignancies occurring in the brain ) and they account for approximately 1.4  % of all cancers [ 1 , 2 ] .
the incidence of brain and central nervous system tumours worldwide in 2008 was 237,913 new reported cases and is projected to increase to 357,377 by 2030 . depending on the grade of malignancy , the current standard treatment includes surgery , chemo- and/or radiotherapy . despite advances in anti - tumour therapy ,
one of the challenges in treating brain tumours is the presence of the blood brain barrier ( bbb ) that limits the access of systemically administered drugs into the tumour site . due to the bbb , lack of lymphatic drainage and lack of professional antigen presenting cells ,
, this concept has been revised , as immune cells have been shown to infiltrate brain tumours   and play important roles in both anti - tumour immunity   and tumour progression [ 5 , 6 ] .
natural killer ( nk ) cells are large granular lymphocytes of the innate immune system .
nk cells can also recognise the fc part of antibodies via low affinity fcriiia ( cd16 ) receptor and perform antibody dependent cellular cytotoxicity ( adcc ) of antibody - coated cells .
moreover , they secrete various cytokines and chemokines , such as interferon gamma ( ifn- ) .
the recognition of target cells and activation of nk cells is controlled by a balance between activating and inhibitory signals mediated by binding of the ligands expressed on target cells to the receptors expressed on nk cells [ 9 , 10 ] .
the inhibitory receptors include nkg2a   and inhibitory killer immunoglobulin - like receptors ( kirs ) [ 12 , 13 ] that recognise major histocompatibility complex ( mhc ) class i molecules , which are often down regulated on virus - infected or tumour cells .
the activating signals are mediated by natural cytotoxicity receptors ( ncrs )   and nkg2d   that recognise ligands expressed on infected or transformed cells , but absent on most normal cells .
some kir receptors also mediate activating signals , however , they engage the mhc class i ligands with lower affinity than inhibitory kirs . upon activation ,
nk cells induce apoptosis of target cells by secreting granules containing perforin and granzymes   or by signalling via death receptors .
nk cells play an important role in anti - tumour immunity as several lines of evidence link nk cell surveillance , anti - tumour activity   and prognostic significance [ 2024 ] in various types of cancers .
the potential of nk cells as effectors against brain tumours has been demonstrated in vitro [ 25 , 26 ] and in vivo [ 27 , 28 , 29 ] .
however , translating these promising results into the clinic requires answers to several important questions related to patients immune status : ( 1 ) do nk cells infiltrate brain tumours and if yes , are they functional ?
( 2 ) what is the prognostic significance of nk cells activity and their infiltration into the tumour ? ( 3 ) how do the tumour cells escape nk cell - mediated surveillance and killing ?
clinical trials using nk cells against brain tumours have already been conducted   and some are in progress or recently completed ( for example clinicaltrials.gov : nct00823524 and nct00909558 ) . moreover ,
other experimental therapies might be influenced by patients nk cell activity   and also several treatment strategies have been shown to stimulate nk cell - mediated lysis of tumour cells [ 8 , 27 , 28 , 32 ] .
therefore , it is of great importance to take into consideration the brain tumour patients immune status and encourage further research in this direction .
our review summarizes what is already known in this field and what aspects need further investigation .
we also provide an overview of pre - clinical studies conducted and discuss the clinical application of nk cell - based therapy .
numerous studies have investigated the immune system of brain tumour patients , mainly in the context of malignant glioma .
brain tumour patients suffer from extensive immunosuppression due to lymphopenia , decreased lymphocyte proliferation [ 33 , 34 ] and diminished cytotoxic activity , reduced mhc class i expression on monocytes [ 33 , 34 ] and predominance of anti - inflammatory t - helper 2-type ( th2 ) cytokine production . in one of the first studies , servadei et .
al analysed cytotoxic functionality of peripheral blood lymphocytes isolated from anaplastic glioma patients and compared with those from bladder and kidney cancer patients and healthy donors .
in all three types of cancers they observed decreased adcc and decreased spontaneous lymphocyte mediated cytotoxicity ( slmc ) when comparing to healthy donors , to the greatest extent in anaplastic glioma patients .
reports investigating nk cell numbers and activity in the peripheral blood of glioma patients show conflicting results ( reviewed in dix et al . .
the functionality of circulating nk cells is often affected in brain tumour patients and it may result from immunosuppressive factors released by tumour cells .
an example is transforming growth factor - beta ( tgf ) that down - regulates the expression of nkg2d activating receptor on nk cells isolated from glioblastoma patients compared to those from meningioma patients .
moreover , decreased absolute numbers of nk cells have been observed in the blood of glioblastoma patients receiving concomitant radiation therapy and temozolomide .
the remaining question was whether the nk cells infiltrate the tumour site . in 1988 , stevens et al . characterised the immune infiltrates in gliomas , carcinoma metastases , craniopharyngiomas and meningiomas . to identify nk cells within the brain tumour tissue , the immunohistochemistry ( ihc ) technique with leu7 ( cd57 ) and leu11b ( cd16 ) antibodies
they observed that in most glioma cases nk cells were absent , while carcinoma metastases and craniopharyngiomas were more frequently infiltrated by nk cells .
nk cells constituted a minor fraction among all immune cells infiltrating the tumour , leading the authors to conclude that nk cells do not play an important role in anti - tumour immunity in brain cancer .
similar results were obtained by vaquero et al . for brain metastases   with use of the iot-10 antibody that also recognises cd57 .
in addition , they showed that the degree of nk cell infiltration varied depending on the origin of metastasis . in a recently published work ,
the same group reported no correlation of the degree of cd57 + nk cells with the clinical outcome of patients with brain metastasis .
the critical point of all those studies is that the cd57 marker is expressed only on a subset of nk cells and is also expressed on a significant fraction of t cells .
therefore a more precise analysis is needed to determine the true frequency and degree of nk cell infiltration in brain tumours.table  1nk cell infiltration in intracranial tumours : summary of reviewed literatureauthorstumour typemethod , antibodiesdegree of nk cell infiltrationdistribution of nk cells in the tumour tissuestevens et al .
gliomas grade ivihc , leu7 and leu11blowwithin perivascular cuffsgliomas grade i  iino infiltrationcarcinoma metastasesintermediatearound blood vessels and within tumour parenchymacraniopharyngiomasintenseno specific distributionmeningiomaslow 
brain metastasesihc , iot-10less than 10  % of tils in most cases ( 39/40)mainly associated with vessels and stromayang et al .
glioblastomasihc , cd56perivascular : intermediate or extensive in app.70  % cases ; intratumoural : intermediate in app .
meningiomasflow cytometry , nk cell population defined as cd3-cd19-cd56 + 0.2    0.3  % of all cells 
 ihc immunohistochemistry , til tumour - infiltrating lymphocytes ,  no data reported nk cell infiltration in intracranial tumours : summary of reviewed literature 
 ihc immunohistochemistry , til tumour - infiltrating lymphocytes ,  no data reported contrasting results were reported by yang et al .   in the most recently published work investigating immune cell infiltrates in glioblastomas and comparing it with pilocytic astrocytomas . according to their report
, nk cells frequently infiltrated the glioblastomas , whereas this infiltration was negligible in pilocytic astrocytomas .
, the authors concluded that nk cells may play an important role in anti - tumour immune responses in glioblastoma patients . in this
this method alone might give inaccurate results as cd56 is also expressed by some cytotoxic t cells . according to our observations ,
nk cells were one of the least numerous immune cell populations of all tumour infiltrating immune cells in glioblastomas ( 2.11  %    0.54 , mean    sem ) and were predominantly cd56cd16 .
these results are based on multicolour flow cytometric phenotyping of patients glioblastoma ( gbm ) tumour biopsies .
however , they evaluated the expression of leu11b ( cd16 ) that is expressed only on the major subpopulation of nk cells . in the light of the observations made by stevens et .
al , who did not detect leu11b positive cells despite the presence of leu7 ( cd56 ) positive infiltrates in various brain tumour specimens and our own studies that did not detect cd16 positive nk cells in gbms , it is possible that in oligodendrogliomas a similar expression pattern of cd56cd16 occurs as in the other gliomas .
the most recent study focusing on meningiomas was conducted by domingues et al .   and demonstrated the presence of nk cells within the tumour .
, , nk cells were one of the least numerous immune cell populations infiltrating the tumour .
a number of in vitro and in vivo functional studies have been performed in order to investigate the role of nk cells in anti - tumour immunity in brain cancers and the potential of using them as a therapeutic agent .
investigated the therapeutic efficacy of a toll - like receptor 9 ( tlr9 ) ligand , cpg - oligodeoxynucleotides ( cpg - odn ) , in vivo in a murine glioma model .
they showed that nk cell numbers in brain , blood and spleen decreased with tumour growth , possibly as a result of tumour - induced immunosuppression .
however , they also demonstrated that the therapy they used against glioma induced host immune responses and nk cells mediated the resistance to tumour re - challenge .
another group demonstrated increased cytotoxic activity of splenic nk cells isolated from glioblastoma - bearing animals treated with recombinant adeno - associated virus 2 encoding il-12 .
dendritic cell ( dc ) vaccination has also been shown to stimulate ifn secretion by nk cells and increase their number in the peripheral blood in gbm patients .
on the other hand , alvarez - breckenridge et al . showed in vivo , that nk cells can negatively influence virotherapy against glioblastoma .
evaluated the susceptibility of the glioblastoma stem - like cells to nk cell - mediated lysis in vitro .
they found that both allogeneic and autologous activated nk cells were able to efficiently kill the gbm cells .
compared the gbm cells cultured under serum - free conditions with those serum - cultured in a series of cytotoxicity assays using activated nk cells and t cells as effectors .
they reported that gbm stem - like cells were more susceptible to both nk cell- and t cell - mediated lysis .
moreover , they showed that in combination with the therapeutic antibody cetuximab , nk cells were able to kill gbm stem - like cells via adcc .
our team recently demonstrated that combination treatment with nk cells + mab9.2.27 against the ng2/cspg4 proteoglycan diminished tumor growth that was associated with reduced tumor proliferation , increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls .
therapeutic adcc was mediated by recruitment of ccr2low macrophages into the tumor microenvironment , increased ed1 and mhc class ii expression on microglia that rendered them competent for gbm antigen presentation , as well as elevated ifn- and tnf- levels in the cerebrospinal fluid compared to controls .
the advantages and relevance of these studies were the use of purified nk cells and patient - derived gbm cells .
the prognostic significance of nk cells activity has been demonstrated in patients with various solid tumours [ 2022 , 24 ] . a higher level of cd57 positive cells infiltration into the tumour correlates with better survival of patients with oesophageal squamous cell carcinoma , squamous cell lung cancer   and gastric carcinoma . as mentioned before , cd57 is expressed also on a subset of t cells , therefore very likely both nk cells and t cells contribute to better patient outcomes . in the study of kondo et al .
, a high activity of peripheral nk cells positively correlated with longer survival of colon cancer patients .
they also observed that cumulative 5-year metastasis - free rates were higher in the group of patients with high nk cell infiltration ( 90 vs. 60  % ) .
moreover , tumour cells have been shown to express ligands for activating receptors expressed on nk cells [ 50 , 51 ] , making them potentially susceptible to nk cell - mediated lysis .
various immunotherapies against brain cancer have been tested so far ( reviewed in ) , including dc vaccination , adoptive t cell transfer , and transfer of lymphokine activated killer ( lak ) cells .
most of the clinical trials referring to nk cell activity utilized autologous lak cells combined with il-2 injections ( recently reviewed in .
lak cells are obtained from peripheral blood mononuclear cells ( pbmcs ) cultured at conditions that stimulate their expansion and activation .
the final product is a mixture of t cells ( majority of them are cytotoxic t lymphocytes ( ctls ) and nk cells .
those trials demonstrated partial efficacy and moderate adverse effects ( mostly edema ) [ 54 , 55 ] .
the weak point of those studies is that the effector cell populations contributing to anti - glioma effect have not been characterised and the mechanisms of treatments tested have not been investigated .
however , they showed the potential of cellular therapy against the most malignant brain tumour .
therefore , using pure nk cells against solid tumours becomes an attractive alternative strategy to using lak cells . to date , only one phase i clinical trial has been conducted , where pure autologous nk cells were used as effector cells against brain tumours . in this study , 9 patients with recurrent malignant glioma received intracranial or intravenous injection of nk cells expanded from patients pbmcs . in 4 of 9 patients ,
the authors concluded their therapy was less toxic but was as efficient as lak cells combined with il-2 .
the limited efficacy of experimental therapies utilizing autologous lak or nk cells might be due to the local and highly integrated tumour - induced immune suppression and immune escape mechanisms .
ctls require antigen presentation and co - stimulation signals that are often impaired in brain tumour patients .
our group has also demonstrated that gbm cells highly express mhc class i molecules , that are ligands for inhibitory receptors expressed on nk cells .
therefore , engaging nk cells to kill the tumour cells might require additional , combination treatments that will enhance their activity and/or overcome tumour immune escape mechanisms .
one possibility is the use of tumour specific antibodies that would induce adcc mediated by engaging the fcriiia receptor ( cd16 ) on nk cells ( fig . 
an interesting approach investigated in other solid tumours is the use of fusion proteins comprised of a part recognising the tumour - specific antigen conjugated to a ligand for activating nk cell receptors , like nkg2d ( fig . 
however , these two strategies are of limited use in the highly heterogeneous brain tumours .
there rarely exist in these malignancies tumour - specific antigens that are expressed on all the tumour cells in each patient and that are also absent on the normal cells .
moreover , it may be envisaged that these tumours would eventually escape the treatment by developing antigen loss variants .
reducing nk cell inhibition mediated by interactions of inhibitory kir receptors with human leukocyte antigen ( hla ) ligands may be achieved by blocking kir receptors with specific antibodies ( fig . 
another alternative solution would be the use of allogeneic nk cells instead of patients own cells ( fig . 
nk cells have been shown to mediate the graft versus leukemia ( gvl ) effect in haematological malignancies .
the hypothesized mechanism is the receptor - ligand mismatch between the kir receptors expressed on donor s nk cells and cognate hla ligands expressed by recipient s cells resulting in the absence of inhibitory signals mediated by kir receptors expressed by a subpopulation of nk cells .
moreover , the patient s normal cells are spared from killing due to the absence of activating ligands that are expressed almost exclusively on transformed cells .
we hypothesise that the same strategy could be used in the treatment of solid tumours , including brain cancer .
moreover , a combination of autologous or allogeneic nk cell - based cellular therapy with immune stimulating treatment such as inhibitory kir - blocking antibody could give better results compared to each of those strategies applied alone .
brain tumour patients could also benefit from combining adoptive transfer of nk cells with already approved therapeutic antibodies such as cetuximab , an anti - epidermal growth factor receptor ( egfr ) antibody .
figure  2 summarizes the potential of combination approaches and the role of nk cells in anti - tumour immune response.fig . 
a therapeutic antibodies , such as cetuximab ( anti - egfr antibody ) can induce antibody - dependent cellular cytotoxicity ( adcc ) mediated by fcriiia receptor ( cd16 ) expressed on nk cells .
b immunoligand is a fusion protein : the part recognising specific antigen is conjugated to ligand for activating receptor .
immunoligand binds tumour - specific antigen and stimulates nk cells ( endogenous and/or transferred , both autologous and allogeneic ) via interaction with activating receptor ( e.g. nkg2d ) .
c applying kir - blocking antibody reduces the inhibition of both endogenous nk cells as well as adoptively transferred autologous nk cells .
lack of recognition of tumour s hla by inhibitory kir receptors results in the absence of inhibitory signals and nk cell activation .
+ activating receptor , l ligandfig .  2the interplay of nk and other immune cells in anti - tumour responses .
endogenous and/or adoptively transferred autologous nk cells can be stimulated by immunoligands ( 1 ) and/or kir blocking antibodies ( 2 ) .
therapeutic antibodies such as cetuximab ( anti - egfr antibody ) can induce antibody - dependent cellular cytotoxicity ( adcc ) ( 3 ) .
other possible strategy is cellular therapy with allogeneic nk cells with kir - hla mismatch approach ( graft vs. tumour effect , gvt ) ( 4 ) . stimulated and/or alloreactive nk cells are able to directly kill the tumour cells , as well as to secrete pro - inflammatory cytokines such as ifn to further stimulate other immune cells : macrophages , microglia ( 5 ) and t cells ( 6 ) .
nk cells may play an important role in dc vaccination due to dc - nk cell cross - talk further stimulating anti - tumour immune response ( 7 ) . stimulated macrophages , microglia and dc
can present tumour - associated antigens ( 8) and induce ctls - mediated cytotoxicity ( 9 ) .
+ activating receptor , l : ligand treatment strategies for using nk cells against brain tumours . a therapeutic antibodies , such as cetuximab ( anti - egfr antibody ) can induce antibody - dependent cellular cytotoxicity ( adcc ) mediated by fcriiia receptor ( cd16 ) expressed on nk cells .
b immunoligand is a fusion protein : the part recognising specific antigen is conjugated to ligand for activating receptor .
immunoligand binds tumour - specific antigen and stimulates nk cells ( endogenous and/or transferred , both autologous and allogeneic ) via interaction with activating receptor ( e.g. nkg2d ) .
c applying kir - blocking antibody reduces the inhibition of both endogenous nk cells as well as adoptively transferred autologous nk cells .
lack of recognition of tumour s hla by inhibitory kir receptors results in the absence of inhibitory signals and nk cell activation . + activating receptor , l ligand the interplay of nk and other immune cells in anti - tumour responses .
endogenous and/or adoptively transferred autologous nk cells can be stimulated by immunoligands ( 1 ) and/or kir blocking antibodies ( 2 ) .
therapeutic antibodies such as cetuximab ( anti - egfr antibody ) can induce antibody - dependent cellular cytotoxicity ( adcc ) ( 3 ) .
other possible strategy is cellular therapy with allogeneic nk cells with kir - hla mismatch approach ( graft vs. tumour effect , gvt ) ( 4 ) . stimulated and/or alloreactive nk cells are able to directly kill the tumour cells , as well as to secrete pro - inflammatory cytokines such as ifn to further stimulate other immune cells : macrophages , microglia ( 5 ) and t cells ( 6 ) .
nk cells may play an important role in dc vaccination due to dc - nk cell cross - talk further stimulating anti - tumour immune response ( 7 ) . stimulated macrophages , microglia and dc
can present tumour - associated antigens ( 8) and induce ctls - mediated cytotoxicity ( 9 ) .
the nk cells have limited access into the nascent brain   and they are the least abundant immune cell population within the brain tumour microenvironment . however , different methods used to detect nk cells in the tumour tissue led to conflicting results .
recent developments give an opportunity to study the infiltration of nk cells with more precise methods , such as with nk cell - specific antibodies or with multicolour flow cytometry with a panel of markers .
it is also important to reveal the mechanism of nk cell recruitment , as various experimental therapeutic agents have been demonstrated to stimulate nk cell - mediated anti - tumour immune responses [ 8 , 27 , 28 , 32 ] .
nk cells might also influence the efficacy of other anti - cancer treatments , therefore their translation into clinic requires knowledge about the patients immunological status .
malignant brain tumours develop multiple mechanisms of immune escape and both local , and systemic immunosuppression [ 33 , 36 , 45 , 61 , 62 ] .
many immunotherapies are based on patients own immune cells isolated from peripheral blood . therefore , it is of great importance to investigate the functionality of autologous nk cells in brain tumour patients and the tumour - derived factors that could negatively influence nk cell - based therapy .
so far , very few studies have been conducted and most of them investigated whole lymphocyte populations .
research focused on pure nk cell fractions could facilitate the development of efficient treatment strategies directly utilizing nk cells as anti - cancer agents or stimulating endogenous nk cells . to summarize
, nk cells play an important role not only in anti - tumour immunity but they are potent effectors that may be considered for developing novel immunotherapies . however , stringent research is required to determine the impact of nk cell infiltration into the tumour site and their therapeutic efficacy in brain tumour patients . investigating nk cells in the context of brain tumour is also essential for improving the nk cell - based immunotherapies that are increasingly investigated for clinical development for brain tumour patients .
 natural killer ( nk ) cells are lymphocytes that play an important role in anti - tumour immunity . their potential against brain cancer has been demonstrated in vitro and in vivo , both as a direct anti - tumour agent and in experimental therapies stimulating endogenous nk cell cytotoxicity . 
 however , the clinical translation of these promising results requires detailed knowledge about the immune status of brain tumour patients , with focus on the nk cell population . in this report 
 , we provide an overview of the studies investigating nk cell infiltration into the tumour , emphasizing the need of revision of the methodologies and further research in this field . 
 we also discuss the potential of using autologous or allogeneic nk cells as effector cells in cellular therapy against brain cancer and developing immunotherapies stimulating endogenous nk cell - mediated anti - tumour response , such as blocking inhibitory killer immunoglobulin - like receptors . 
 combination of nk cell adoptive transfer with targeted therapies , such as anti - egfr therapeutic antibody ( cetuximab ) could also be a potent strategy . 
Introduction: why focus on natural killer cells for brain tumours?
What is the immune status of brain tumour patients?
Do NK cells infiltrate the brain tumour?
Are NK cells a potent anti-tumour agent against brain cancer? Functional studies
Can the anti-tumour potential of NK cells be exploited in the clinic?
Conclusions and perspectives: why do we need further investigation in this field?