cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries .
advancements have been made in traditional treatment modalities that have been used for decades , namely , surgery , radiotherapy , and chemotherapy .
in addition , with the value of early diagnosis in cancer therapy recognized , the technology of early diagnosis is also advancing .
although these treatment modalities play an important role , the results are not entirely adequate , especially for advanced cancers .
cancer is still a major public health problem worldwide , and new treatment modalities and strategies are still needed to optimize patient outcomes .
cancer immunotherapy , which can be generally classified as passive or active , has always been an attractive and potentially efficient treatment for cancer patients .
passive immunotherapy , consisting of infusion of donor t lymphocytes and transfer of anticancer monoclonal antibodies , has been proven to be an effective treatment for a variety of cancers [ 3 , 4 ] , and continued advances in t - cell engineering and antibody should further enhance their clinical impact .
however , vaccines , which represent active immunotherapy , are based on the manipulation of the host immune system to fight cancer and provide a path to obtain long - lasting responses in cancer patients .
as one of the major players in active immunity , cytotoxic t lymphocytes ( ctls ) play a critical role in immunity against cancers .
a variety of vaccine strategies have been designed to meet this goal , and recent phase ii / iii clinical trials using these vaccines have achieved promising results .
cancer vaccines enhance the antitumor immune response by providing the early signals of activity ; dendritic cells ( dcs ) play an important role in this immune response activation , which involves a number of complex processes .
first , dcs must capture tumor antigens , process the captured antigen for presentation on major histocompatibility complex ( mhc ) molecules ( either class i or class ii ) , and then migrate to draining lymph nodes . if capture and processing is accompanied by a suitable activation signal , dcs will enhance the activation of the immune response . if not , dcs will instead induce tolerance .
second , in lymphoid organs , tumor - antigen - loaded dcs are capable of triggering protective t - cell responses , especially ctl responses . in this process ,
dcs require a maturation signal ( i.e. , a stimulatory adjuvant ) in order to elicit the desired ctls [ 9 , 10 ] . without a maturation signal , dcs present antigens in a stable state , which promotes tolerance by inducing regulatory t cell ( treg ) production and thereby thwarting an antitumor response [ 1113 ] .
in addition , the ability of dcs to promote a ctl response also depends on the interaction of a positive t - cell costimulatory molecule ( i.e. , cd28 , ox40 ) with dc surface receptors ( i.e. , cd80/cd86 , ox40l ) ; the interaction of the negative t - cell costimulatory molecule ( i.e. , ctla-4 , lag-3 ) with the dc surface receptors can limit the activity of ctls by promoting treg formation . for these reasons ,
the appropriate utilization and regulation of dcs correlate with the success or failure of vaccine design .
all in all , increasing immune activation by dcs is a critical step for improving ctl antitumor immune responses ( figure 1 ) .
targeted therapy is a typical representative of selective , mechanism - based therapy and has become a new treatment option .
based on the molecular mechanisms of ctl activation , targeted drugs can not only promote the capture of tumor antigens by dcs can also enhance the expression of costimulatory molecules such as cd40 , cd80 , and cd86 on the dc surface .
moreover , targeted drugs block important inhibitory signals for activated t - cells , thereby maintaining t cell activation and potentiating tumor destruction .
in general , through regulation of dcs and activated t cells , targeted drugs effectively boost ctl activation [ 1517 ] .
furthermore , activated ctls must enter the tumor microenvironment to perform their functions , at which point a variety of negative regulatory signals suppress the immune response ; ctls must overcome the tolerance induced by tumor cells in order to mediate their cytotoxic effects .
therefore , the effects of drugs that target the processes that generate ctl antitumor immune responses can be complementary and synergistic with cancer vaccines .
combinatorial therapy provides a new treatment modality that enhances the vaccine - induced ctl antitumor immune response .
however , greater focus on the appropriate dosage , sequencing , and timing of the targeted drugs is needed , as it will probably be crucial to the success of combinatorial strategies .
another key issue for combinatorial therapies is the toxicity of these regimens . in general , the ultimate goal of enhancing vaccine - induced ctl antitumor immune responses
encouraged by positive preclinical and clinical data [ 2125 ] , the research into the appropriate utilization and regulation of dcs in vaccine design continues , and the study of the combination of vaccines and targeted drugs aims to solve the above - mentioned problems .
dcs , as the most efficient antigen - presenting cells ( apcs ) , have been increasingly used in various strategies of vaccine design to enhance their unique ability to activate antitumor ctls .
these strategies facilitate the capture of tumor antigens by dcs , which helps to stimulate the activation of ctls in the next step . in the past few decades
, vaccine design strategy has included several antigen - loading techniques to activate dcs , such as ( i ) pulsing dcs with peptides or proteins , ( ii ) transfecting dcs with dna or rna , ( iii ) loading dcs with tumor - cell lysates or tumor cells , and ( iv ) infecting dcs with bacterial , viral , or yeast vectors .
( figure 1(a ) ) in contrast , vaccines that do not utilize dcs tend to depend on the immunogenicity of the vaccines itself , which are not enough to elicit a stronger ctl response [ 27 , 28 ] .
dcs transfected with the human telomerase reverse transcriptase ( htert)-hil18 gene were capable of eliciting a stronger htert - specific ctl response in vitro than those stimulated with the htert construct only .
this strategy of vaccine design utilizes dc activation in vitro in order to avoid the complex regulation of dc exposure to antigen in vivo . on antigen encounter in vivo ,
dcs require a suitable activation and maturation signal to promote immunity ( enhanced capture , processing and presentation of tumor antigen - derived peptides ) rather than tolerance [ 7 , 30 ] .
these signals include endogenous tumor cell lysates ( e.g. , high mobility group proteins or atp ) and exogenous molecules ( e.g. , toll - like receptor ( tlr ) ligands or antibodies ) .
like tlr ligands ( which will be discussed later in this paper ) , monoclonal antibodies can been used to facilitate the capture of tumor antigens by dcs .
trastuzumab and cetuximab are clinically efficacious monoclonal antibodies that are directed toward the tumor - associated receptor tyrosine kinases her2 and egfr , respectively .
these antibodies not only downregulate oncogenic signaling , but also enhance the induction of tumor - specific ctls [ 31 , 32 ] .
these antibodies coat tumor - associated receptors on tumor cells and bind fc receptors expressed on dcs simultaneously to form immune complexes that contribute to the capture of tumor antigens by dcs .
further , fc receptor - mediated opsonization enhances the expression of costimulatory molecules ( i.e. , cd40 , cd80 , and cd86 ) on the dc surface , boosting t - cell activation .
therefore , direct inoculation of patients with vaccines that activate dcs in vivo requires combining this targeted therapy in order to promote a desired immunogenic phenotype .
confirmed that combining a cancer cell vaccine and her2 antibodies resulted in significantly longer survival rates than either therapy alone in a transgenic mouse model of breast cancer .
this may be due to the fact that the combination therapy increased the capture of the cancer vaccine by dcs , enhanced the expression of costimulatory molecules ( i.e. , cd40 , cd80 , and cd86 ) on the dcs , and elicited greater tumor - specific t - cell responses .
recently , a phase i / ii clinical trial of trastuzumab combined with a her2 peptide vaccine showed that 69% of patients developed ctl immune responses to tumor cells that expressed her2 .
similarly , the egfr antibody cetuximab also enhances dc - mediated phagocytosis , increases the expression of the mhc class ii molecules , costimulatory molecules on dcs and induces a highly efficient antigen - specific ctl response in vitro   ( figure 1(b ) ) .
tumor - antigen - loaded dcs are capable of triggering protective t - cell responses , especially by activating the ctl response in lymphoid organs . however , without a maturation signal ( i.e. , a stimulatory adjuvant ) , dcs can not elicit the desired ctl response . when dcs present antigens in a stable state without an immunogenic maturation signal , regulatory t cells ( treg ) are induced , which both promote tolerance of antitumor responses and directly counters the antitumor response .
what 's more , at the same time , stimulatory adjuvant can induce costimulatory molecules ( i.e. , cd40 , cd80 , and cd86 ) on the surface of dcs and the release of cytokines ( i.e. , ifn- , ifn- , and il-12 ) by dcs , which are required for t - cell activation and differentiation ( figure 1(c ) ) .
many initial attempts at cancer vaccines lacked a consideration of this immune process , specifically the requirement of dcs for a stimulatory adjuvant in order to induce the desired ctls .
peptide - based vaccines that were used to treat thousands of patients often lacked an effective dc - activating adjuvant . on the one hand ,
free peptides may be rapidly cleared before being loaded onto dcs because of their poor pharmacokinetic properties , so their half - lives may also be short . on the other hand , without an adjuvant , dcs might remain in the steady state and gradually promote tolerance rather than immunity . as a result ,
the ctl response to the selected tumor antigens is poor and therapeutic benefit is minimal . because the function and importance of dcs in stimulating ctl responses is well known now , effective dc - activating adjuvants are increasingly being used in vitro and in vivo . for example
, il-2 is an important stimulatory adjuvant for maintaining dc growth and has received fda approval for use in melanoma and renal cell cancer .
granulocyte - macrophage colony - stimulating factor ( gm - csf ) is necessary for the growth and differentiation of dcs , and tumor - necrosis factor-  ( tnf- ) can promote dc maturation and antigen - presenting capabilities [ 38 , 39 ] .
tlr ligands are the ideal adjuvants , allowing a variety of functions required for ctl activation .
secondly , tlr ligands induce the expression of costimulatory molecules ( cd40 , cd80 , and cd86 ) on dcs that are required for t - cell activation .
thirdly , tlr - induced cytokines , principally il-12 , ifn- , and ifn- , guide t - cell differentiation toward either cd8 + cytotoxic lymphocytes or cd4 + t helper type 1 ( th1 ) cells .
several tlr ligands have shown significant promise for the treatment of cancer for example , the tlr7 agonist imiquimod that is currently approved for treatment of superficial basal cell carcinoma .
although monotherapy with tlr agonists may provide a benefit in certain types of cancer , our interest is centered on the role of tlr agonists as stimulatory adjuvants .
encouraging results have been reported in human melanoma with an antitumor peptide vaccine using cpg - odn ( a tlr agonist ) as an adjuvant , which promotes the generation of rapid and strong ctl antitumor responses .
researchers also confirm that tlr agonists facilitate vaccine - induced ctl responses in many different mouse tumor models .
therefore , its use as a vaccine adjuvant is perhaps the most extensively explored application for tlr agonists .
  after capture , processing and presentation of tumor antigens by dcs and activation of ctls , activated ctls must enter the tumor microenvironment to perform their functions ; at this point , a variety of negative regulatory signals interact and form a network to resist the immune response .
these negative regulatory signals mainly originate from tumor cells   and stromal cells such as immune cells , inflammatory cells , endothelial cells , and fibroblasts .
tumor - induced mechanisms of immunosuppression are as follows : ( i ) tumor cells expansion or enabling the local accumulation of treg cells , which release suppressive cytokines and serve to silence immune responses [ 47 , 48 ] , ( ii ) tumor cells also encourage the formation of myeloid - derived suppressor cells ( mdscs ) , which use indoleamine 2,3-dioxygenase ( ido ) released by tumor cells to silence the responses of cytotoxic cd8 + t cells and helper cd4 + t cells while simultaneously promoting the formation of treg cells , and ( iii ) tumor cells secrete a variety of products including the above - mentioned ido , vascular endothelial growth factor ( vegf ) and surface molecules such as pd - l1 .
vegf promotes the expansion of mdscs , blocks the maturation of dcs , and inhibits the expression of costimulatory molecules [ 50 , 51 ] .
pd - l1 engages receptors on the surfaces of activated t cells and causes t - cell anergy or exhaustion [ 52 , 53 ] ( figure 2(a ) ) .
in addition , t cells express other receptors that negatively regulate the immune response in the tumor microenvironment .
for example , ctla-4 is a well - studied key negative regulator . when dcs present peptide epitopes to the t - cell receptor ( tcr ) and simultaneously present b7 costimulatory molecules ( cd80 , cd86 ) to cd28 on specific antitumor t cells , these t cells become activated .
ctla-4 is subsequently upregulated and competitively engages b7 to attenuate t - cell responses   ( figure 2(b ) ) .
lag-3 is another negative receptor on the t cell surface that not only limits the activity of cd8 + and cd4 + t cells but also augments the activity of treg cells [ 55 , 56 ] .
moreover , macrophages polarize to type 2 macrophages ( m2 ) because of hypoxia in the tumor microenvironment .
m2-like macrophages secrete and release cytokines such as ccl17 , ccl22 , or ccl24 , which recruit treg cells to the local environment . however , there are still a large number of immunosuppressive mechanisms in the tumor microenvironment which are not mentioned in this paper
.   thus , even if pathologically induced auto - ctls could avoid negative regulatory signals and overcome diverse immunosuppressive networks within the tumor microenvironment , they would still be too weak to promote tumor destruction .
although vaccines ( e.g. , sipuleucel - t ) can be used to increase the number of ctls , immunosuppression still occurs in the tumor microenvironment and can attenuate vaccine - induced ctls .
for this reason , a larger number of targeted drugs have been designed and used in clinical trials to counteract negative regulatory mechanisms .
imatinib can decrease ido expression and lead to an increased ratio of ctls to treg cells in a mouse model of gastrointestinal stromal tumors .
reported that imatinib has a direct inhibitory effect on treg cells , including decreased numbers of treg cells and weak immunosuppressive capacity .
the combination of imatinib plus a dc vaccine resulted in decreased numbers of treg cells , increased t - cell - derived ifn  production and fewer metastases compared with either therapy alone in a bcrabl lymphoma model .
the targeted drug sunitinib , a multityrosine kinase inhibitor that blocks vegfr function , decreased the function and number of mdscs in the tumor microenvironment of a mouse model of colon cancer .
consequently , sunitinib inhibited the number and function of  treg  cells and augmented the activation of cytotoxic cd8 + t cells and helper cd4 + t cells .
furthermore , sunitinib diminished the expression of pd-1 , pd - l1 , and ctla-4 .
the combination of a dc vaccine plus sunitinib prolonged survival compared with either agent alone in a b16 melanoma model .
the anti - ctla-4 antibody ipilimumab has recently been approved by the fda for use as a first - line or second - line therapy in patients with advanced melanoma .
previous studies have confirmed the fundamental importance of ctla-4 in controlling t - cell function , and ctla-4 ligation is also important for the immune suppressive function of treg cells [ 62 , 63 ] .
reported clinical trial data that led to the fda approval of anti - ctla-4 for the treatment of metastatic melanoma .
their data indicates that ipilimumab , most notably , achieves durable benefits for more than 2.5 years .
a subsequent clinical trial confirmed that ipilimumab plus standard care improved overall survival compared with standard care alone ( 11.2 months versus 9.1 months ) and significantly increased the proportion of surviving patients for at least 3 years of followup ( 20.8% versus 12.2% ) .
as the most important function of vaccine - induced ctls , ctl - mediated cytotoxic effects play a critical role in immunity against cancers .
ctls have the ability to kill tumor cells following the interaction of their tcr with a specific mhc - i complex on tumor cells .
this process is mainly mediated by the ability of effector ctls to release perforin , granzyme , and tnf- , or to express fas ligand [ 66 , 67 ] .
cytotoxicity proceeds through a multistep mechanism : ( i ) the tcr combines with a specific mhc - i complex on tumor cells , and then the remodeling of the actin cytoskeleton , reorganization of the cell surface , and repositioning of cytoplasmic proteins result in the formation of a so - called immune synapse , ( ii ) polarization of the microtubule organizing center ( mtoc ) toward the target occurs , and lytic granules move along the microtubules toward the mtoc , and ( iii ) lysis occurs through the polarized release of the content of cytotoxic granules ( a process also referred to as degranulation ) or expression of fas ligand on the ctl surface .
the lytic pathway uses mainly two types of mechanisms to kill target cells : one is perforin mediated and another is fas ligand ( fasl ) based . the perforin - mediated mechanism is believed to involve secreted perforin , which forms  pores  in the target membrane . then co - secreted granzymes pass through the  pores ,  enter cells and activate a cascade of caspases including caspase-3 and caspase-8 [ 7072 ] .
the fasl - based mechanism involves crosslinking of the cell surface death receptor fas expressed on target cells induced by cell surface fasl expressed on ctls .
cross - linked fas rapidly induces the assembly of an intracellular  death - inducing signaling complex  ( disc ) , which recruits and activates caspase-8 and ultimately activates caspase-3 , which serves as a common substrate for these two killing mechanisms [ 7275 ] . both of these mechanisms eventually induce apoptosis of target cells . although ctls have the capacity to efficiently kill tumor cells , tumor cells may develop escape mechanisms to evade ctl - mediated killing .
for instance , tumor cells acquire resistance to perforin and granzyme or alter the expression of death receptors . in addition
, the genetic instability of tumor cells can lead to the alteration of proapoptotic regulators , one of the most important being p53 .
it has been demonstrated that the p53 status of tumor cells has a key role in determining the fate of the antitumor ctl response , because it regulates fas receptor expression , cellular flice / caspase-8 inhibitory protein ( cflip ) short protein degradation , and cd95-mediated apoptosis .
in addition , tumor cells can lower the expression of mhc class i molecules to avoid detection or increase the expression of antiapoptotic proteins in order to resist cytotoxic mechanisms .
therefore , the combination of vaccines and some targeted drugs may counteract these escape mechanisms and improve ctl - mediated tumor destruction .
( figure 3 ) in contrast to the escape mechanism described above , some targeted drugs enhanced sensitization of tumor cells to ctl - mediated destruction . for example , the proteasome inhibitor bortezomib sensitizes tumor cells toward adoptive ctl attack .
a preclinical trial of bortezomib plus vaccination with dna encoding a tumor - specific protein is planned . the combination of an hpv e7-based vaccine and bortezomib prolonged the steady state of the disease , in contrast to either therapy alone , which had minimal effects . the increased survival correlated with enhanced sensitivity of the bortezomib - treated tumor cells to killing by e7-specific ctls .
the possible mechanisms are as follows : ( i ) bortezomib may enhance the baseline activity of granzyme and caspase-8 in tumor cells , which could provide sensitization towards apoptosis , ( ii ) bortezomib may sensitize tumor cells to ctl - mediated lysis through enhanced expression of noxa , a bh3 protein that sequesters potent antiapoptotic proteins such as myeloid cell leukemia sequence 1 ( mcl1 ) , or ( iii ) bortezomib may increase tumor cell surface expression of the tnf - related apoptosis - inducing ligand receptor dr5 and fas , which further augments nk cell cytotoxicity .
in addition , some evidence suggests that bortezomib can also sensitize tumor cells to nk cell - mediated lysis by downregulating peptide loading and mhc class i molecule expression .
this is due to bortezomib inhibiting the proteasome that generates peptide epitopes for mhc class i molecules , rendering tumor cells more susceptible to killing by nk cells .
pi3 k - akt inhibitors are another class of targeted drugs that sensitize tumor cells to ctl - mediated destruction through pi3 k - akt pathway inhibition ; this eliminates some prosurvival signals such as expression of antiapoptotic proteins , thereby increasing tumor cell lysis by perforin and granzymes released from ctls [ 83 , 84 ] .
confirmed that tumor cells in which akt can be inducibly expressed showed enhanced resistance to ctl killing upon akt activation compared with noninduced cells .
the enhanced resistance involved an increase in mcl1 expression , which is an antiapoptotic protein .
demonstrated that a tumor cell line selected for resistance to vaccination therapy upregulated akt compared with wild - type cells that are sensitive to vaccination therapy .
this result was associated with increased levels of bcl-2 , ciap1 , and ciap2 , which are also antiapoptotic proteins . moreover , noh et al . demonstrated that inhibiting the upregulation of the akt pathway increased the ctl - mediated killing of akt - upregulated tumor cells in vitro and akt inhibition in combination with vaccine - augmented ctl responses .
a variety of strategies have been used to design vaccines that can induce a durable and long - lasting host immune response to fight cancer . however , a series of processes are necessary in order for a vaccine - induced ctl immune response to play a positive role in destroying cancer . in any step in these processes
even when ctls are activated , negative signals causing t - cell anergy or exhaustion may occur in the tumor microenvironment .
ctl - mediated cytotoxic effects also are affected by the escape mechanisms of tumor cells . for these reasons , when designing vaccines , we attempt to utilize and regulate dcs to elicit a much more potent ctl immune response .
this strategy includes antigen - loading techniques in vitro , supplying targeted drugs that facilitate the capture of tumor antigens by dcs in vivo , and using stimulatory adjuvants that promote the whole development and function of dcs .
in addition , targeted drugs can be used which block important immunosuppressive signals in the tumor microenvironment , and overcome the tolerance of tumor cells .
the strategy of combining vaccines and targeted drugs increases ctl - mediated tumor cell lysis . in the context of advances in the understanding of how immunity , immunosuppression and tolerance
regulate antitumor immune responses , immunotherapy and targeted drugs might have complementary and synergistic roles in cancer treatment ; therefore , the combination of vaccines and targeted drugs may become a more accepted practice in cancer treatment .
 vaccine - induced cytotoxic t lymphocytes ( ctls ) play a critical role in adaptive immunity against cancers . 
 an important goal of current vaccine research is to induce durable and long - lasting functional ctls that can mediate cytotoxic effects on tumor cells . to attain this goal , there are four distinct steps that must be achieved . to initiate a vaccine - induced ctl antitumor immune response , dendritic cells ( dcs ) 
 must capture antigens derived from exogenous tumor vaccines in vivo or autologous dcs directly loaded in vitro with tumor antigens must be injected . 
 next , tumor - antigen - loaded dcs must activate ctls in lymphoid organs . 
 subsequently , activated ctls must enter the tumor microenvironment to perform their functions , at which point a variety of negative regulatory signals suppress the immune response . 
 finally , ctl - mediated cytotoxic effects must overcome the tolerance induced by tumor cells . 
 each step is a complex process that may be impeded in many ways . 
 however , if these steps happen under appropriate regulation , the vaccine - induced ctl antitumor immune response will be more successful . 
 for this reason , we should gain a better understanding of the basic mechanisms that govern the immune response . 
 this paper , based on the steps necessary to induce an immune response , discusses current strategies for enhancing vaccine - induced ctl antitumor immune responses . 
1. Introduction
2. Facilitating the Capture of Tumor Antigens by Dendritic Cells
3. Stimulating the Maturity of Dendritic Cells and Enhancing the Activation of T Cells
4. Enhancing the Effect of Vaccine-Induced CTL Antitumor Immune Responses in the Tumor Microenvironment
5. Overcoming the Resistance of Tumor Cells to CTL-Mediated Cytotoxic Effects
6. Conclusions