haptens are small molecules that elicit an immune response when bound to a carrier protein . haptens have been used to boost immune responses to antigens , to study acd and ibd , and to induce autoimmune responses , viral wart regression , and even antitumor immunity . for years , haptenated protein ( bovine serum albumin ( bsa ) or ovalbumin ( ova ) ) was mainly utilized to induce strong immune responses in animal models to help unravel the basics of t- and b - cell - mediated responses . paul et al .   immunized bsa - tolerized rabbits with dnp - modified bsa producing antibodies to the dinitrophenyl ( dnp)-bsa conjugate , bsa alone , and dnp alone , suggesting potential cross - reactive responses . classically , b - cells are known to recognize the dnp - bsa conjugates via membrane bound igm , process them , make antibody against the dnp , and present the bsa to cd4 + t - cells . these abilities of haptens have made them a tantalizing molecule for use in several settings . haptens have been widely used to induce chs , the animal model of acd , a type iv delayed hypersensitivity reaction that is one of the most prevalent skin diseases in the world [ 3 , 4 ] . phase where the hapten is applied to skin for the first time , followed by an  elicitation phase where the hapten is applied to a different skin area of the animal [ 35 ] . an in - depth analysis of the innate and adaptive immunologic mechanisms of chs and acd is covered in three recent reviews by martin et al . , in this review , we will briefly cover these immune reactions to allow for a general understanding of how these reactions may apply to antitumor immunity . when a drug is metabolized , its metabolites can form potent haptens , which bind self - protein and sometimes elicit autoimmune responses [ 7 , 8 ] . hapten - carrier conjugates have been used in the past as drug - abuse therapies [ 9 , 10 ] , inducing an immune response against the drug of interest . haptens have also been used to create autoimmune models in mice , such as ibd [ 1117 ] , and to cause viral wart regression via epifocal hapten application [ 18 , 19 ] . the ability of haptens to cause autoimmunity and wart regression is an important concept to consider when applying the use of haptens to cancer immunotherapy setting , as the immune response to cancer is similar to an autoimmune response . indeed , haptens have been tested as a treatment of cancer several times in the past . in this review , we examine the four main concepts of hapten - mediated antitumor treatment : ( 1 ) ex vivo haptenation [ 2131 ] , ( 2 ) in situ haptenation [ 32 , 33 ] , ( 3 ) epifocal hapten application [ 3442 ] , and ( 4 ) antigen - hapten administration [ 4347 ] . despite the wealth of experiments in this field , the mechanisms underlying these treatment approaches are largely unclear and require further study . we attempt to give a critical analysis of the use of haptens to induce tumor regression and suggest studies that must be done to fill the large knowledge gaps and further the field . haptens are < 1  kda in size and elicit an immune response when bound to a carrier protein , including tolerized antigen . haptens are not immunogenic by themselves , as they are too small to be recognized by the immune system . most haptens are electrophilic compounds that covalently bind to nucleophilic residues creating new antigenic epitopes ; an exception to this would be metal ions functioning as haptens . most haptens act as cutaneous allergens , eliciting acd - like reaction on the skin . the most common haptens are urushiol ( the toxin in poison ivy ) , fluorescein , nickel , oxazolone ( ox ) , dnp , and phosphorylcholine . each hapten has a unique property that determines its allergenicity in terms of how quickly the hapten binds , how readily it can permeate the skin , and its electrophilicity , hydrophobicity , and subsequent bioavailability . contact hypersensitivity is usually measured through ear swelling , as the secondary challenge application is on the ear ( elicitation phase ) ; this is the widely used method to confirm sensitization of a hapten and unravel the immune mechanisms of haptens and chs . the body of literature on haptens and chs reveals the use of several different animal models and haptens that lead to conflicting explanations of a certain step in the immune pathology of chs , which should be considered when creating a general mechanism of chs . while outlining our understanding of the mechanisms of chs , we primarily focus on the aspects that will be important for hapten - mediated tumor regression . the sensitization phase is when a hapten is first applied to the skin of an animal , typically the shaved abdomen , to prime the immune system toward the hapten . figure 1 summarizes some of the cells and mechanisms thought to be involved in this priming event . upon application to the skin , haptens immediately interact with keratinocytes ( kc ) , langerhans cells ( lc ) , and dermal dendritic cells ( ddc ) . hapten binding to kcs causes them to release il-1 , il-18 , tnf , and gm - csf , activating lcs and ddcs and inducing their migration to the draining lymph node where they mature and present hapten - antigen to nave t - cells [ 46 , 4852 ] . dinitrofluorobenzene ( dnfb ) application to dermal dendritic cells in vitro upregulates mapk and cd40 , a coactivation signal for antigen - presenting cells ( apcs ) and t - cells . haptenation also causes the release of  danger signals ,  such as hyaluronic acid ( ha ) , extracellular matrix ligands for toll - like receptors , such as extradomain a+ fibronectin containing extra type iii domain a ( fneda+ ) , prostaglandin e2 ( pge2 ) , reactive oxygen species ( ros ) , heparin sulfate , tenascin , b defensins , and fibrinogen [ 4 , 5 , 54 ] , from haptenated cells , which play an integral role in innate immune activation . for instance , blocking ha degradation significantly reduces chs sensitization , while the release of pge2 activates lcs and induces their migration . the in vitro formation of ros in dcs is thought to cause degradation of the extracellular matrix , creating endogenous ligands for toll like receptors ( tlrs)-2 and -4 , as well as nucleotide - binding oligomerization domain ( nod ) like receptors ( nlrs ) [ 4 , 6 ] . keratinocytes are mainly stimulated by nlr - dependent mechanisms , specifically nlr family , pyrin domain containing 3 ( nlrp3 ) . nlrp3 stimulation triggers caspase-1 activation , which causes the release of il-1 and il-18 from keratinocytes and apcs . this nlr - dependent pathway is stimulated by adenosine triphosphate ( atp ) efflux from haptenated and subsequently damaged cells . atp binds to the purinergic receptor , p2rx7 , a ligand gated ion channel that allows the release of k+ from the cell and provides further innate activation signals for lcs and ddcs , helping them mature . langerhans cells play a pivotal but controversial role in the sensitization phase ; when lcs are depleted , the ear - swelling responses are reduced . further , lcs and ddcs work together to initiate chs sensitization [ 56 , 57 ] . the role of the lcs seem to be area and time of depletion dependent , for instance , lcs had a larger role in the flank than in the ear and lc depletion three days prior to hapten priming did not impair chs but lc depletion 1 day prior did impair chs [ 58 , 59 ] . it was shown that only ddcs , not lcs , migrate to the draining lymph node ( dln ) to activate and stimulate hapten - specific t - cells [ 52 , 60 ] . despite this controversy , initial reports using mast cell deficient mice through a c - kit mutation showed that chs was enhanced , although this is hard to interpret as c - kit mutation affects many cells [ 4 , 60 ] . diphtheria toxin - induced mast cell - deficient mice had reduced chs and t - cell priming [ 4 , 61 , 62 ] . mast cells also stimulate ddcs via intercellular adhesion molecule-1 ( icam-1 ) or leukocyte function - associated antigen-1 ( lfa-1 ) and tnf [ 4 , 61 , 62 ] . mast cells and dendritic cells are critical during the sensitization phase , causing dc migration and maturation [ 4 , 5 , 61 , 62 ] . upon maturation by keratinocyte stimulation , the dermal apcs activate naive t - cells and invariant natural killer t ( inkt ) cells by presenting the haptenated antigen ( peptide and lipid ) via mhci / ii or cd1d , respectively . peptide presentation depends on whether the haptenated protein becomes internalized and processed via the endosomal compartments , followed by mhc - i presentation , or whether the haptenated proteins are on the extracellular surface and cross presented via mhc - i to cd8-t - cells . many haptens enter the cells through passive diffusion and bind to intracellular proteins , which are presented by mhc - i , h-2k , to naive cd8 + t - cells . presentation to naive t - cells leads to the formation of hapten - specific memory t - cells with the capability to become hapten - specific effector t - cells ( cd4 + and cd8 + ) . thus , these effector cells cause damage and regulate immune responses at the elicitation site [ 4 , 5 ] . haptenation also causes the release of endogenous glycolipids that are processed and presented via cd1d to inkt cells in the liver . in balb / c and cba / j mice inkt - cells become stimulated within 30 minutes via  stimulatory  lipids in the liver and release il-4 [ 6568 ] . the il-4 , along with haptenated antigen in the circulation [ 66 , 67 , 69 ] , stimulates naive b-1 cells within 1 hour to migrate to the draining lymph node and form  cs - initiating b-1 cells ,  a distinct class of b-1 cell , that creates hapten - specific igm [ 70 , 71 ] . in c57bl/6 mice , however , these inkt - cells have an inhibitory role   as they release il-4 and il-13 which , along with t - regulatory cells that release il-10 , suppress the formation and function of the hapten - specific memory t - cells [ 73 , 74 ] . the differences in function of inkt - cells are most likely because balb / c mice have a more th2-based immune response , whereas c57bl/6 mice have a more th1-like immune response . paust et al .   showed that natural killer ( nk ) cells induced chs reactions in rag mice ( devoid of t- and b - cells ) . further experimentation   showed that liver nk cells are able to transfer chs to naive animals in 1 hour . currently , there is no literature on how these nk cells become activated , although one can infer that nk cells are more likely to become activated due to a lack of engagement of inhibitory receptors . ly49c , found on these hapten - specific nk cells , is specific for h-2k binding . if the self - protein being presented is haptenated , it may no longer appropriately recognize or bind with the ly49c , causing nk cells to recognize the cell as foreign . it is likely that dnp - bound mhc will affect ly49c binding , but this requires experimental verification . in summary , after hapten application , keratinocytes stimulate dapc maturation and migration , leading to activation of hapten - specific memory t - cells , inkt - cells , cs - initiating b-1 cells , and hepatic nk cells . the sensitization phase appropriately primes the immune system to the hapten , so that the elicitation phase can occur quickly and with optimal immune response . upon secondary hapten challenge early  and  late  events , resulting in swelling and severe damage of the challenged area . the early elicitation phase which peaks within 2 hours of challenge and dissipates by 4 hours seems to be antigen - independent , while the late elicitation phase occurs within 24 hours of the challenge and seems to be antigen - dependent . each of these concepts needs to be considered for understanding hapten - induced tumor - immunity . figure 2 outlines the steps in the early elicitation phase . upon hapten - challenge , there is antigen - nonspecific inflammation ; inkt - cells are restimulated by the stimulatory lipids released in the liver , causing them to once again produce il-4 . this release causes the restimulation of cs - initiating b-1 cells to produce igm against hapten . the hapten - specific igm and haptenated antigen will go into circulation , form complexes and activate complement c5a [ 65 , 69 , 80 ] through the classical complement pathway . the c5a will then bind to mast cells in the dermis , causing release of serotonin , tnf , and cxcl2 . . in combination with these neutrophils , tnf and serotonin production by mast cells will cause the release of cxcl-10 , ccl1 , 2 , and 5 from the surrounding tissue and the upregulation of icam-1 , e- and p - selectin on endothelial cells in the vasculature , leading to hapten - specific t - cell recruitment [ 4 , 61 , 62 , 81 ] . neutrophils are also brought to the area by the release of cxcl1 and 2 from keratinocytes after hapten - challenge and elicit t - cell infiltration [ 4 , 82 ] . fasl and perforin expression of neutrophils is essential to initiate proper t - cell infiltration , as administration of soluble fasl in the challenge area had similar responses . keratinocytes are known to release proinflammatory cytokines ( il-1 and tnf ) upon hapten stimulation , causing vascular endothelial cells to upregulate icam-1 and p- and e - selectins . in the absence of il-1 and tnf , chs is suppressed . keratinocytes also produce many chemokines that allow for hapten - specific t - cell entry into the challenged area , the most important being cxcl10 , which will be bound by the cxcr3 on th1 cells . figure 3 outlines the steps in the late elicitation phase , which occurs within 24 hours of hapten - challenge . ddcs , lcs , kcs , and endothelial cells process haptenated antigen as previously described and present the antigen to hapten memory t - cells that have migrated to the dermis during the early elicitation phase . once stimulated in the dermis , memory t - cells will form hapten - specific cd4 + and cd8 + t - cells . typically , inkt cells can either play a stimulatory or inhibitory role that depends on the mouse model used to study inkt cells , c57bl/6 mice versus cba / j mice , respectively . in cba / j mice , inkt cells can release ifn that helps to promote cd8 + effector development when working in conjunction with  t - cells [ 65 , 87 ] . in c57bl/6 mice , the inkt - cells release il-4 and il-13 , which suppress chs reactions , possibly by stimulating a th2 response . this is in contrast to other strains of mice wherein il-4 release helps to stimulate cs initiating b-1 cells . t - cells seem to  collaborate  with inkt - cells to elicit cd8 + t - cell - mediated damage during chs . upon adoptive transfer with these two cell subtypes , there was a strong ear swelling response at 2 and 24 hours post - dnfb challenge , but if either one was depleted , the ear swelling significantly decreased . this collaboration of inkt - cells and  t - cells helps to activate  tcr+ cs - effector cells . langerhans cells , once thought to be the main apc of haptenated - ag , are thought to have more of a regulatory role in the elicitation of chs . depletion of epidermal lcs in hapten - sensitized mice elicited greater chs responses   as lcs can suppress chs responses via cd40-cd40l interactions with cd4 + t - cells causing the release of lc derived il-10 . notably , lcs tolerize cd8 + t - cells by activating foxp3 + t - regulatory cells ( t - regs ) in mice sensitized with a weak hapten and then challenged with a strong hapten . it is likely that ddcs , endothelial cells , and kcs , not lcs , present antigen to memory t - cells in the dermis during the elicitation phase [ 5 , 92 ] . hapten - specific t - cells will traffic to the elicitation site by upregulation of chemokines , selectins , and adhesion molecules and differentiate into their appropriate effector or helper status by a multitude of cytokine signals ( from the tissue and activated t - cells ) and haptenated - antigen presentation [ 4 , 5 , 92 ] . honda et al .   summarizes the roles of different cytokines in the elicitation phase of chs and the large difference between the reactions elicited with the haptens trinitrochlorobenzene ( tncb ) , ox , dnfb , and fluorescein isothiocyanate ( fitc ) , all which are known to be th1 haptens except for fitc , which is known to be a th2 hapten . they further emphasize that the differing effect of cytokines reported in the literature is due to the hapten , animal model , and possibly even the microbiota of the animals in the specific animal facility . we think that haptenation of microbiota will release multiple danger signals , haptenated bacterial proteins , and haptenated bacterial lipid , which can uniquely stimulate different types of chs reactions through various innate immune responses , inkt cell responses , and t - cell responses . the  hapten atopy hypothesis ,  proposed by mcfadden et al . , states that haptens delivered a few times by epifocal application will stimulate tlr4 through danger signal release , leading to a th1 immune response , but repeated and prolonged exposure to haptens will likely shift the response from th1 to th2 . when tlr4 is stimulated , it will weakly upregulate tlr2 expression to drive th2 responses , possibly by heat - shock protein ligand upregulation . the repeated exposure of the haptens and weak stimulation of tlr2 will form th2 cytokines , which will downregulate th1 cytokines and suppress tlr4 function . indirectly support this hypothesis by showing that different types of hapten challenges , acute ( one challenge ) , subacute ( three challenges ) , and chronic ( 513 challenges ) result in different chs responses . in the chronic exposure versus acute exposure , there is a decrease of th1 cytokines ( tnf , inf , il-2 , and il-12 ) , an increase of th2 cytokines ( il-4 , il-5 , and il-13 ) , and an increase in t - regulatory cytokines ( il-10 ) , indirectly giving support to the  hapten atopy hypothesis  . there are multiple different t - cell subsets that are involved in the elicitation of chs - related cellular damage . classic delayed - type hypersensitivity is cd4 + regulated , and for many years it was assumed that chs worked the same way . now it is evident that both cd8 + and cd4 + t - cell subsets are involved in eliciting chs . .   showed that cd8 + effector t - cells were the main cells that elicited chs damage and cd4 + effector t - cells minimally acted as chs effectors . along with this notion , hapten - specific cd4 + t - cells are thought to consist of more cd4 + t - regs than effector cells , each having their own effect on chs responses , inhibitory and stimulatory , respectively . it is likely that both cd4 + and cd8 + effector t - cells work in tandem to elicit damage , as shown in cd4 + and cd8 + t - cell ko mice experiments where both subsets had great impact on chs responses . it seems that cd8 + t - cells are the main chs - effector t - cells , and that cd4 + t - cells have a dual role , eliciting minimally the effector function and largely the regulatory function . cd8 + t - cells elicit damage in the haptenated area during chs elicitation phase by augmenting cytotoxicity with perforin and fas / fasl interactions . cd8 + t - cells have also been shown to release ifn and il-17 , which can stimulate neutrophils to draw more cd8 + t - cells to the area by keratinocyte - induced upregulation of chemokines [ 83 , 100 ] . il-17 release seems to play an important role in chs and acd [ 101 , 102 ] , as th1/th17 cells infiltrate acd areas upon niso4 application in human patients . these results found in chs and acd models show that cd8 + t - cells and possibly th17 cells are crucial players in chs reactions . t - regulatory cells down - regulate contact hypersensitivity by using the il-2 produced from hapten - specific cd8 + effector cells . chs - associated t - regs traffic to the inflamed site during the elicitation phase   and likely inhibit chs by ctla-4 and cd86 interactions between t - regs and cd8 + t - cells , as treatment with anti - ctla-4 antibody increased chs responses . they also inhibit chs by il-10 release , which is known to suppress chs   and block entry of hapten - specific effector t - cells into the challenge site . taken together , t - regs play a large role in chs regulation and are important when considering hapten - induced tumor regression . extensive studies were performed by hans ulrich weltzien 's group from 1992 to 1997 looking at the tcr specificities of cd4 + and cd8 + t - cells and the way in which haptenated protein is presented to t - cell receptors ( tcrs ) . they showed that trinitrobenzene sulfonic acid ( tnbs)-like haptens are h-2k restricted ; haptenated ag can be processed intracellular in the er / golgi to be presented by mhc i , and trinitrophenyl ( tnp)-specific t - cell clones were able to recognize haptenated and unhaptenated portions of designed tryptic fragments of tnp - octapeptides . tnp - specific cd4 + t - cell clones were able to recognize many different tnp - modified peptides , as long as tnp was present . these papers suggest the ability of hapten - specific cd8 + clones to recognize unhaptenated portions of amino acid chains , whereas hapten - specific cd4 + t - cells only recognize haptenated protein . 
  t - cells and inkt - cells were shown to work together to release ifn , which would stimulate a tc1/th1- like response ; however , they were shown to inhibit chs reactions during elicitation by hindering the development of hapten - specific cd8 + t - cells . t - cells played a role in eliciting dinitrochlorobenzene ( dncb)-induced chs in lambs . , presented at  the american association of immunologists conferences in may of 2013 ,  focuses on the dermal  t - cells in terms of how their depletion suppresses chs reactions . it seems that il-17 dermal  t - cells are important in inducing chs reactions . the involvement of dermal  t - cells during elicitation is unclear and needs further study . first described by o'leary et al .   and paust et al . , chs was induced in a rag mouse ( lacking b- and t - cells ) with the assumption being that no ear swelling would be seen ; these animals got an ear swelling reaction close to normal . using adoptive transfer systems , it was seen that these nk cells were hepatic , expressed thy-1 , ly49c , and cxcr6 and could elicit chs responses 4 months after sensitization . l- , p- , and e - selectins and nkg2d were found to play an important role in nk - mediated chs reactions [ 75 , 76 ] . ,   who looked at a distinct cd3 , cd16 , perforin+ , cd56 , cd16 , and cd62l ( noncirculating ) nk cell populations that produced ifn and tnf in nickel - challenged acd regions of humans . unexpectedly , these nk cells did not elicit a memory - like response as previously described but did contribute to keratinocyte apoptosis ; this could be a mouse versus human phenomena . confirmed the presence of nk cell - mediated chs in mice devoid of b- and t - cells , although the response was markedly diminished compared to wild - type ( wt ) mice . these cells produced ifn , ifn , and il-12 , were thy1 + and mac1 + ( fully licensed ) , cxcr6-dependent , and could elicit a chs reaction in as little as 1 hour after transfer from a sensitized to nave animal . did a comparison of t - cell - mediated to the nk cell - mediated reactions using the  classical  chs protocol with dnfb ; they showed that the nk cells failed to create a genuine chs response in rag2 mice , as the dnfb ear challenge did not require sensitization to elicit an ear swelling response . 's   observations by performing similar adoptive transfer experiments of nk cells and showed that the responses were similar to transferred cd8 + t - cells . however , the recall response of these transferred nk cells upon a second hapten challenge was much weaker and short - lived than that of transferred cd8 + t - cells and there was little cd45.1 + t - cell infiltration into the challenged site in the nk cell - transferred mice . it seems as though nk cells play some sort of role in chs , although they may only be able to elicit true chs reactions in adoptive transfer settings and may only help to elicit damage at the haptenation site . there are many common allergens that cause chs : metals likes nickel or gold , certain antibiotics like neomycin , topical anesthetics , natural compounds such as urushiol , the irritant in poison ivy , and many more . these all act directly as haptens , inducing a chs - like reaction in the skin . there are instances where metabolizing a drug or chemical can lead to autoimmune - like responses , idiosyncratic drug reactions . this is when a drug 's metabolite acts as a hapten and binds to cellular proteins , eliciting an immune response and antibody production to the metabolite - protein conjugate , the metabolite alone , and the protein alone . these drugs are prohaptens , or chemicals that are not protein - reactive unless metabolically activated to the electrophilic state . a common example of this is penicillin - induced hemolytic anemia . when the penicillin enters the body , it is metabolized in the liver and forms penicillenic acid , similar to the hapten oxazolone , which then covalently binds to red blood cells ( rbcs ) . antibodies ( igg ) can form against the hapten - coated rbcs , which are then killed by antibody - dependent cellular cytotoxicity ( adcc ) and cleared by macrophage opsonization . hydralazine , a hypertension drug , is known to cause drug - induced lupus ( dil ) through its metabolites . it was seen that hydrogen peroxide and other oxidants from the lungs react with hydralazine to produce metabolites that bind to self - protein . about 5% of the patients who take this drug develop dil - like symptoms [ 130 , 131 ] . there are several other examples of drug - induced autoimmunity in several different contexts , most involving the binding of a drug or its metabolite to self - protein inducing antibody production . in all cases , the drug or metabolite acts as a hapten to induce autoimmunity . paul et al .   showed proof of principle experiments that haptens could allow the immune system to overcome peripheral tolerance . by injection of haptenated - bsa , bsa - tolerized rabbits were able to induce the production of antibody towards the hapten , the bsa , and the conjugate . haptens have been shown to induce hapten - specific cd8 + t - cell cross - recognition of haptenated and unhaptenated octapeptides as previously described . kang et al .   showed hapten - mediated autoimmunity experimentally in hen egg lysozyme ( hel)-transgenic ( tg ) c57bl/6 ( b6 ) mice that were immunized with hel or hapten - modified ( phosphorylcholine [ pc ] ) hel ( pc - hel ) . hen egg lysozyme immunization failed to induce antibody responses against hel in the transgenic animals , but the pc - hel generated large amounts of anti - hel antibody . this break in tolerance was by t - cells seen through t- and b - cell depletion and adoptive - transfer experiments . lastly , pc - hel was better at generating hel epitopes for t - cell recognition following antigen processing . they suggest that the  generation of new immunogenic epitopes of self - antigens may result in breaking self - tolerance and lead to the production of autoantibodies  . despite these examples , none of these papers showed the ability of these reactions to induce immune damage , as this would be indicative of autoimmune disease . experimentally induced autoimmunity seems to be a hapten - dependent reaction that does not occur in the absence of the hapten . the unique property of haptens to induce immune reactions against self - peptide has been utilized in many other settings besides chs . haptens have been commonly used to induce acute and chronic ibd in rats and mice using the haptens 2,4,6-trinitrobenzene sulfonic acid or 2,4-dinitriobenzene sulfonic acid ( dnbs ) to induce immune reactions in the intestine [ 1115 ] . te velde et al .   reviewed the models of tnbs - induced ibd , clearly stating many of the problems present in the field . ibd reactions seem to be hapten - dependent , and the hapten does not induce autoimmune reactions to the intestine once it is out of the animals ' system . ennifar et al . applied for a patent   for a novel hapten - carrier conjugate that stimulates the production of antibodies against nicotine . these antibodies could be used to treat nicotine - addicted patients , as they passively lower the nicotine levels in the serum and brain . a similar idea was tried using a novel hapten - conjugate , 6-glutarylmorphine - keyhole limpet hemocyanin ( klh ) , conjugate that induced antibodies against morphine and heroin in rats . the treatment increased rat movement and attenuated other drug - induced behaviors , compared to the control group , in morphine and heroin addicted rats ; this was associated with antibodies against the morphine and heroin . these methods have not been extensively studied , making long - term dependence on the haptens unclear . clearly , haptens have been used in many contexts to study certain diseases and induce responses against certain malignancies . the properties of haptens to induce reactions are fascinating , although it seems as though these reactions may be hapten - dependent , and many will wane as the hapten is cleared . despite this , the ability of haptens to induce reactions against self - protein , even if haptenated , is a unique property that make haptens tantalizing targets for cancer immunotherapy . in the following sections , we will review how haptens have been used to treat tumors , their advantages and disadvantages , the challenges present in the field , and possible directions of study to further the field . the use of haptens to induce tumor regression is not a new one , as many groups have attempted several different methods of hapten - mediated tumor regression . ( 1 ) the tumor is removed , haptenated ex vivo , and injected back into sensitized animals or patients [ 2131 ] . ( 2 ) the tumor is haptenated in situ ( in the tumor ) [ 32 , 33 ] . ( 3 ) the tumor area is haptenated epifocally ( at the tumor site ) to induce a chs - like reaction [ 3442 ] . to note , this method has only been utilized for cutaneous skin cancers that can invade the epidermis or dermis , as chs reactions require these . ( 4 ) adcc reactions at the tumor site can be induced by intraperitoneal ( i.p . ) or subcutaneous ( s.c . ) administration of antigen - hapten conjugates in mice and patients , respectively with antigen - receptor high tumors [ 4347 ] . these concepts ( table 1 ) , the problems and holes present , and our interpretation of the possible antitumor mechanisms occurring are reviewed below . many groups have utilized ex vivo haptenation to induce tumor regression in mice and humans . were the first group to use ex vivo haptenation as a cancer immunotherapy in mice . they used x5563 cells , a plasmacytoma cell line syngeneic to c3h / hen mice previously shown to generate  killer t - cell activity without inducing helper t - cell activity against tumor - associated transplantation antigen ( tata ) and still grow . they immunized mice with hapten - modified x5563 cells to amplify helper t - cell activity , and augment killer t - cell responses to the tata . they primed mice intraperitoneal ( i.p . ) with trinitrophenyl ( tnp)-bound mouse gamma globulin ( mgg ) in order to generate tnp - specific t - cells . six weeks later , they immunized mice i.p . with tnp - bound x5563 tumor cells , using tnbs , generating killer t - cells against x5563 and tnp - x5563 tumor cells ; this did not occur in mice primed with unhaptenated tumors . they further amplified this response with a pretreatment of tnp - d - gl to ablate tnp - suppressor cell activity . mice were given the full treatment ( tnp - d - gl pretreatment , three days after tnp - mgg immunization , six weeks after immunized i.p . with tnp - x5563 cells once a week for five weeks ) and then given a lethal dose of the x5563 cells . the tumor growth was greatly decreased and the mean survival time of the mice increased by 10 days posttreatment . this study only examined the tumor growth for 15 days , so it is likely that the tumor was able to proliferate and grow at further time points . this system is a nice proof of principle but has very little clinical application because it is a lengthy prophylactic treatment that minimally delays tumor growth and the effect of this treatment on an established tumor was not studied . regardless of this , they showed that modification of tata with hapten - induced tnp - reactive helper t - cells , which could amplify killer t - cell generation , resulting in slowed tumor growth and an antitumor immune response in vivo . .   took hamaoka 's model and applied it to a balb / c - lstra leukemia tumor system . they primed mice with tnp - d - gl , three days later , immunized mice with tnp - mgg , and six weeks later , i.p . syngeneic t - cells were stimulated in vitro by co - culturing them with tnp - lstra cells for five days . the tnp - primed splenocytes were collected , mixed with viable lstra cells to perform in vivo tumor neutralization assays by intra - dermally ( i.d . ) injecting the mixture into tnp - sensitized balb / c mice . this stopped tumor formation for at least 11 days after inoculation . despite not showing the effect of this treatment on tumor cell challenges or established tumors , this study highlights the proof of a principle that anti - tumor immune responses can be generated with ex vivo haptenation of tumor cells . investigated ex vivo tnp - modification , using tnbs , of regressor and progressor tumors to cause tumor rejection of unmodified progressive tumor cell lines in mice . they created a system of tumor inoculation rejection in c3h / hen mice using primary s.c . immunization of tnp - bound 1591 regressor fibrosarcomas , followed 28 days later by a secondary immunization of a tnp - bound 3152 progresser fibrosarcoma and tertiary challenge of unmodified-3152 progressor cells . the resistance to progressor tumor cells was adoptively transferred with total splenocytes to nave animals . by antibody depletion , it was seen that lyt-1 - 2 + t - cells and lyt-1 + 2- t - cells expressing nonclassical helper t - cell phenotypes elicited the resistance . weak  tumor - associated antigens by tnp - modification , despite the eventual progressor tumor growth . it would be interesting to see what would have happened if they had used a cytotoxic hapten , like tncb for their immunizations , as hapten - mediated cell death may have elicited better immune response , or if they had sensitized the animals to tnp before vaccination , as this may have enhanced the immune response to the haptenated cells . [ 24 , 26 , 28 , 30 ] utilized the ex vivo haptenation as well as in situ haptenation mouse studies by fujiwara et al . [ 32 , 33 ] as the basis for clinical trials using ex vivo tumor cell haptenation as a primary treatment for metastatic melanoma or as an adjuvant treatment after surgical resection of nodal metastases in stages iii and iv metastatic melanoma patients . two weeks before vaccination , patients were pretreated with cyclophosphamide ( cy ) and 2 days later sensitized to 1% dnfb . patients were treated with cy three days before the dnp vaccination ; this was repeated every 28 days . cyclophosphamide has long been known to enhance chs - like responses as it decreases the percentage and number of cd4 + cd25 + t - regs that suppress the induction of chs . the dnp - vaccine was made by surgical resection of primary melanoma , irradiation , modification with dnfb , and intradermal injection back into patients along with bacillus calmette - guerin ( bcg ) , a known cancer immune adjuvant . forty - six patients with measurable metastases were treated , resulting in 20 patients with clinically evident inflammatory responses in nodal , subcutaneous , or intradermal tumors . these tumors had increased cd8 + t - cell infiltration , compared to prevaccination tumors , which strongly expressed hla - dr and cd69 suggesting activation . in addition , 140 t - cells clones were created , 70 of which could kill autologous melanoma cells in vitro . it is commonly seen that tumor - infiltrating lymphocytes ( tils ) are able to kill tumor cells in vitro once stimulated , so it is unclear if the dnp - vaccine was responsible for this cytotoxicity . of the 40 evaluable patients , only five had clinical responses , four complete and one partial , with a median duration of 10 months . one patient remained melanoma free for 10 years after treatment . in the same publication , berd et al . looked at the antitumor effects of dnp vaccination as a postoperative adjuvant therapy ; they compared 41 patients treated with the vaccine after surgical resection to 22 patients who received surgical resection with administration of unhaptenated cells . dnp vaccinations in 4-week intervals and cy was given 3 days before the first 2 vaccinations . the results correlated to a 3-year disease - free survival of 59% for the patients vaccinated with hapten - melanoma cells compared to about 24% for the patients that received the unhaptenated melanoma cells , suggesting that a good clinical response depended on the haptenation of the injected melanoma cells . this was only a short and small study , so it is hard to make concrete conclusions from this , although it indicates that dnp - vaccination is more useful as a postadjuvant therapy with less tumor burden . of note , the control unhaptenated vaccine used in this study was not included for any of the subsequent trials [ 2429 ] . sato et al .   studied the immune response induced by the dnp - modified vaccine in these trials . they collected serum and peripheral blood lymphocytes ( pbl ) from 27 patients before dnfb sensitization ( day 0 ) , after dnfb sensitization ( 2 weeks ) , after two vaccinations ( day 63 ) , after four vaccinations ( day 119 ) , after six vaccinations ( day 175 ) , and after eight vaccinations ( day 231 ) for immunologic study . there were dth responses to dnp - modified autologous pbl and melanoma cells , although dth responses to unmodified cells were not reported . they detected the development of anti - dnp antibody in 24 of 27 patients that was not induced by dnfb sensitization alone . dnp - modified autologous lymphocytes  in vitro ; there was no response to unconjugated or tnp - conjugated autologous lymphocytes . cd8 + and cd4 + t - cells from these stimulated pbl were able to respond to dnp - modified lymphocytes , however , only cd8 + t - cells could respond to dnp - modified melanoma cells . none of these cells were able to respond to unmodified autologous pbl or tnp modified - autologous melanoma cells . these responding cd8 + t - cells produced high amounts of ifn and could kill dnbs - modified autologous melanoma cells ; cytolytic activity to unmodified cells was not examined . in their discussion , the authors mention that they did not see an in vitro reaction to unmodified melanoma cells , but state that their clinical findings still hold true and that there is inflammation of distant tumor sites . they propose that in humans , the majority of t - cells are going to be reactive to dnp - melanoma , but there may be a small subset of cells that are able to react with the unmodified melanoma cells . of note , they showed no reaction of the responder t - cells to unmodified melanoma cells and did not study how these responder cells would specifically respond to modified or unmodified melanoma antigens ( i.e. , gp100 or hmw - maa ) that are known to elicit a t - cell response [ 29 , 136 ] . sato et al .   further observed that the dnp - specific t - cells from patients were able to respond to small dnp - modified peptides associated with the mhc , although these responses were limited to one hplc peptide fraction of autologous melanoma . of note this paper suggests that these t - cells are not going to respond to unmodified melanoma cells , which suggests that the hapten - specific t - cells are not affecting the tumor cells and may not be the only factor in the inflammation of distant metastases as concluded by berd et al . [ 24 , 26 , 28 , 30 ] . in 1997 , berd et al .   used the dnp - vaccine as a postsurgical adjuvant treatment after resection of nodal melanoma metastases in 62 patients . they observed 45% relapse - free survival in stage iii melanoma patients compared to historical controls , stage iii patients from an ecog ifn+ resection study and an ecog resection only study , which showed 34% and 22% , respectively . the hla class i phenotype ( a3+a2 ) , number of metastases ( lower ) , age ( > 50 years old ) , dth to unmodified autologous melanoma , and tumor inflammation seen in patients posttreatment were all positively correlated to overall survival . there were no experiments or discussion of the antitumor mechanism occurring in the patients except for histology of resected tumors posttreatment showing lymphocyte infiltration . the data is difficult to interpret as the controls groups were historical controls , albeit the fact that the inclusion of patients in the trial was based on poor prognosis and no patient was excluded that had extranodal extension of melanoma . however , the results would have been clearer if there had been a control group that only received unhaptenated tumor cells , as done in their earlier trials , to ascertain the importance of the haptenation in eliciting a response . further immunogenic studies are necessary as well as studies with appropriate controls to unravel the efficacy of haptenation . in 2004 , berd et al .   extended the 1997 study to 214 patients with 5-year overall survival of 44% . patients with dth responses to unmodified autologous melanoma had a 5-year overall survival of 59% , double that of the dth - negative group , whereas dth to dnp - modified melanoma gave no overall survival benefit . they retrospectively observed that a baseline skin test with the dnp - vaccine before the start of treatment ( on day 8 and 3 ) acted as an induction dose , which increased the overall survival of patients . as much of the data was based on clinical observations , there was no direct immune correlation between the vaccine and the tumor responses [ 24 , 28 ] . used the dnp - vaccine to treat pulmonary melanoma metastases in 97 stage iv patients . in this study , 11 responses out of 83 evaluable patients , two complete , four partial , and five mixed , were observed . the study describes several case reports of patients who had tumor regression of pulmonary metastases . along with this , only 27 of 83 ( 33% ) patients had tumor inflammation following the dnp - vaccine ; lymphocytes and cd3 + cells infiltrated these tumors . beside this , there were no immune correlates studied in this paper and it is difficult to know whether treatment caused the observed clinical outcome . manne et al .   studied the tcr rearrangement of the associated tils in inflamed melanoma metastases after the dnp - vaccine . they observed that 9 of 10 inflamed tumor samples had dominant peaks in the same v families . however , it was not tested if these tcrs were melanoma antigen - specific or if they could recognize unmodified melanoma cells . the clinical trials using dnp - vaccine [ 24 , 26 , 28 , 30 ] lack immunologic data linking the dnp - vaccine to an immunologic response at unmodified melanoma sites . the main focus of these papers seems to be t - cell responses , when it is now clear that multiple different cell subsets are involved in hapten responses ; nk cells , inkt - cells , mast cells , b-1 cells , and neutrophils should have been considered in this study and could have been causing the distant tumor inflammation they observed . along with this , there was no direct comparison of the dnp - treatment versus same the vaccine without dnp - modification after the first clinical trial , making it hard to know the efficacy of the subsequent trials . lastly , there is no data showing the efficacy of the in vitro haptenation , as it is likely that there were a small percentage of unmodified cells present in the vaccine that could have elicited the inflammation seen in the tumors . extended these clinical trial protocols as a postsurgical adjuvant therapy for 410.1 mammary carcinoma - bearing balb / c mice . injection ( every 10 days for the duration of the experiment ) of either unmodified or dnp - modified , irradiated 410.4 tumor cells with bcg . importantly , the clinical trials by berd 's group injected the vaccine intradermally [ 2430 ] , whereas sojka et al .   injected subcutaneously , which greatly alters the immune responses occurring . the dnp - modified treatment resulted in about 40% relapse - free survival of the mice , while the unmodified treatment was about 20% . they looked at multiple different parameters of the dnp vaccine to see what portions of the treatment were important and to study some immune correlates to the vaccine . relapse - free survival decreased with the depletion of cd4 + or cd8 + t - cells . the draining lymph node cells from mice showed a significant increase of ifn production when given dnp - modified versus unmodified vaccine . lastly , there was a significant decrease in relapse - free survival when neutralizing ifn or tnf. surprisingly , the mice in this study were not sensitized to dnp before immunization , as done in berd et al . 's clinical trials [ 24 , 26 , 28 , 30 ] and shown to be crucial for antitumor responses . this study demonstrates a clear immunologic correlation between the hapten - modified vaccine and relapse - free survival of mice with mammary cancer , but does not fully explain the mechanism of this antitumor immune response . importantly , this model is not representative of the clinical trials as it uses a different injection method than the clinical trials and does not use dnp - sensitization , likely eliciting a different response . the immune responses occurring in ex vivo haptenation that elicit antitumor immunity are dependent on the injection site . injection of dnp - modified cells induced tolerance to chs - like reactions in mice . the anti - tumor immune studies dealt with administration of ex vivo haptenated - cells in three ways , i.p . ( hamaoka et al .   and fujiwara et al . ) , i.d . , ( berd et al . [ 24 , 26 , 28 , 30 ] , sato et al . [ 27 , 29 ] , and manne et al . ) , or s.c . injection , most likely to avoid tolerance and to take advantage of different immune responses . however , much of the mechanisms described below are not supported by experimentation , only by inference . the conjugate would have been taken up and processed , upon which the conjugate - protein would be presented to cd4 + helper t - cells causing cross - activation of both the t - cell and the b - cell . this would have caused the b - cell to produce antibodies against the hapten , the protein , and the conjugate   and would have caused the cd4 + t - cell to elicit hapten - antigen specific responses . it is also possible that the antihapten / antitumor igm and igg bound to haptenated cells , inducing adcc and/or opsonization by macrophages . in terms of the work by hamaoka et al .   and fujiwara et al . , the sensitization would form b - cells specific for the tnp , mgg , and tnp - mgg . upon secondary stimulation with tnp - x5563 , the tnp - specific b - cells would quickly recognize the tnp and produce hapten - specific igm , binding tnp - x5563 cells and allowing for opsonization by macrophages or adcc . this would have produced tnp - modified x5563 tumor antigens that could have been recognized and processed by the hapten - specific b - cells causing further cross - activation and the formation of cd4 + t - cells specific for x5563 cells . these cd4 + t - cells would have likely produced th1 cytokines , like ifn and il-2 , stimulating x5563-specific effector t - cell clones already present in the animal allowing for cytotoxic responses to the tumor . it is also distinctly possible that one of the reasons their treatment was not very effective was because they modified the tumor cells with tnbs , which keeps cells viable . this means that hapten - modified or unmodified protein was not immediately available for b - cells to process and elicit a quick reaction . using a toxic hapten , like tncb , may have made antigen more readily available for b - cells to process due to the tumor cell death . administration of ex vivo haptenated cells is more difficult to interpret as these studies used very different mouse models and delivery systems . when injected into the animal , the regressor tumor cells likely had cytotoxic t - cells that were specific for them and could clear the tumor cells when injected into the animal . if the regressor tumors were tnp - modified , it would have allowed for the release of tnp - bound proteins from these regressor cells that were being actively killed . this would have helped b - cell and cd4 + t - cell cross - activation as described with i.p . injections . upon second immunization , hapten - specific b cells would have recognized the tnp - bound progressor cells and caused cross - activation with cd4 + t - cells , creating b - cells and cd4 + t - cells against the progressor tumor . the activation of tumor specific b - cells would have caused antibody formation against the tumor cells , potentially inducing adcc or opsonization . the cd4 + t - cells would have provided costimulation to cytotoxic t - cells , which are otherwise unable to clear the progressor tumor . these in combination would have likely created the tumor resistance seen in flood et al . as stated above , using a toxic hapten may have made the antigen more readily available for b - cells to process due to the tumor cell death . injection is much different , as it acts as an adjuvant therapy for any established metastases after surgical resection of the primary tumor . importantly , the removal of the tumor could have been the priming step to the immune system as surgical resection of a primary tumor can reverse tumor - induced immunosuppression , even in the presence of metastases . their vaccination protocol killed the cells via irradiation and dnfb modification [ 146 , 148 ] , so it is likely that there would have been much dnp - modified protein available . the actual vaccination protocol probably would have induced a similar response as flood et al . they delivered hapten - modified protein to the immune system , which would have stimulated a strong immune response due to repeated vaccination , hence the enhanced survival of mice with established tumor metastases . the sensitization occurred from dnp - modified tumor cell protein from the first injection , inducing cross activation of b- and cd4 + t - cells as described above and subsequent responses against the tumor . [ 24 , 26 , 28 , 30 ] appears to be the most appropriate ex vivo haptenated - vaccine administration as chs - like immune responses will likely occur . in the clinical trials , patients were mostly sensitized before administration , allowing for the vaccination to induce chs elicitation - like reactions ( table 2 and figure 2 ) . importantly , these reactions will not be as strong as typical chs reactions due to the lack of skin haptenation and subsequent innate immune responses , as the haptenated cells were intradermally injected . the danger signal release from skin haptenation would not have occurred ; meaning restimulation of keratinocytes and dermal apcs would have occurred more slowly , causing less cytokine release . also , no  early  elicitation of chs - initiated mechanisms would have occurred , as inkt - cells specific for haptens would not have become activated , implying that hapten - igm from cs initiating - b-1 cells would not be produced . decreased keratinocyte and cs - initiating b-1 activation would reduce stimulation of mast cells and neutrophils , lowering chemokine , selectin , and adhesion molecule upregulation in the vasculature and the trafficking of hapten - specific t - cells and nk cells to the area . despite this , there would have been involvement of hapten - specific t - cells and hepatic nk cells , as the bcg will cause stimulation of the innate immune system allowing for recognition of haptenated - antigen . bcg likely initiated peripheral immune responses unrelated to the hapten vaccine , which might have slightly inhibited the response , as the immune system could have been  busy  mounting a new response . it may have served berd et al . [ 24 , 26 , 28 , 30 ] to epifocally apply dnfb to the site of the i.d . injection , eliciting a chs reaction that could have exposed the vaccine to the immune system in a chs context . despite all this conjecture , it is hard to know how an antitumor response would have formed as i.d . injection would elicit a hapten - specific immune response and the dnp - vaccine trials did not show much experimental evidence of antitumor immune responses occurring from the vaccination . another important concept to consider is that haptenation in this fashion may not have induced a bystander effect ( kill distant , unmodified tumor cells via immune responses ) since the process seems to be hapten - dependent . much of the justification for the work done was based on weltzien 's group 's papers between 1992 and 1997 , as earlier described [ 63 , 64 , 107 , 108 ] . in this work , they saw the ability of hapten - specific cd8 + t - cell clones to recognize and respond to hapten bound and unbound portions of small tryptic fragments of proteins suggesting some cross - reactivity of the cells . an overarching assumption is that this will be true for naturally processed proteins , like that present in the clinical trial treatments using ex vivo haptenation . sato et al . [ 27 , 29 ] show that dnp - specific tils from dnp - vaccinated patients ( that were not present before vaccination ) were specific for only two peptide fragments from a melanoma peptide library and these fragments had to be dnp - modified . to note , no stimulation occurred with unmodified cells . despite clinical observations of bystander effects , it is very hard to decipher what is occurring since there is not much experimental evidence in support of this claim . as stated before , it is possible that unmodified melanoma cells injected into patients with this vaccine induced an immune response along with the dnp - protein response , leading to tumor inflammation and some antimelanoma immune response . despite all the work done on ex vivo haptenation , as alluded above , there is little experimental evidence to suggest that the vaccination induces direct antitumor effects even though the dnp - vaccine trials show survival impacts in patients . along with that , the treatment is expensive and very time consuming and relies on the removal of a tumor mass , making it an untenable option . seemingly abandoned their ex vivo tumor cell haptenation immunization for in situ haptenation of established tumors . they created a tumor regression model in c3h / hen x5563 plasmacytoma tumor - bearing mice ( dermal ) by intratumoral injection of tncb in tncb sensitized c3h / hen mice . as before , they suggested that haptenation would augment tata helper t - cell responses to generate more powerful killer t - cell responses . they established the following method of tumor regression ; pretreatment of cy , 2 days later tncb sensitization , 5 weeks later implantation of tumor cells , ~6 day after implantation intratumoral injection of 0.15  ml 0.5%  tncb into tumor masses between 7 and 10  mm in diameter . importantly , splenocytes from sensitized mice caused in vitro lysis of tnp - x5563 cells , while unprimed mice splenocytes did not . the spleen cells from tumor - bearing mice , stimulated in vitro with irradiated tnp - x5563 tumor cells , along with the addition of tnp - helper cells , resulted in appreciable augmentation of anti - x5563 cytotoxic t lymphocyte ( ctl ) responses . of the fully treated mice , > 50% of them had complete tumor regression , as measured by the absence of myeloma protein from the blood serum 45 days after treatment . of these animals , 90% of them rejected a secondary tumor challenge of 1/10th the original tumor cells , although the data is not shown . an issue of this study is that 0.15  ml of solution was injected into tumors regardless of their size , meaning that smaller tumors would have increased haptenation and vice versa . it is possible that the animals that responded all had smaller tumors , although this was not recorded or mentioned in the study . large injection volumes could potentially cause the tumor microenvironment to be destroyed , causing tumor cell spillage into the animal . the destruction of tumors sites could have also induced enhanced dnp - tumor reactions by the animal due to better availability of the tumor cells . although this was the first model of in situ haptenation of a tumor and subsequent tumor regression , the mechanism remains unclear . furthered their method by showing secondary challenge and neutralization data as well as repeating it in multiple model tumor systems . they repeated their results in the x5563 system , showing that 4 of 5 mice had tumor regression . myeloma protein was not present in their serum for up to 2 months after regression . they challenged mice with only 10 x5563 cells ( 1/10 of the primary inoculation ) intradermally showing that 11 of 12 of the mice could resist the tumor , compared to 0 of 10 in nave mice or 2 of 10 in surgically resected mice ( this data was not shown in their previous paper ) . conversely , they do not show the tumor growth in these injections and use the word resistance ,  which would imply that the tumors still grew after the challenge , even if the treatment slowed their growth . this is supported by winn assays at low e  :  t ratios that shows slight tumor growth 12 days after secondary tumor challenge . in addition , fujiwara et al .   established tnp - mediated tumor regression in mice with methylcholanthrene ( mca)-induced transplantable tumor cells ( mch-1-a1 ) and mca - induced autochthonous tumors using similar methods . for the inducible system , 11 of 25 of the animals had tumor regression for up to 12 weeks . to note , many of the regressed tumors stayed at a constant size or slowly decreased in size for about 5 weeks after tncb injection , there after dramatically increasing or decreasing in size . the reproducibility of tumor regression in different tumor models is encouraging , but the fact that the secondary tumor challenges were only resisted and not rejected suggests that this method may not induce strong antitumor immune responses and may be hapten - dependent . in situ haptenation offers the most challenging explanation of what occurs , as it relies on the immune cells present inside the tumor microenvironment to elicit responses . it is likely that the haptenation of tumor cells will cause massive amounts of cell death , as typically seen from haptenation , of not only the tumor cells but any of the stromal cells associated with the tumor . this will cause the release of many danger signals and haptenated protein , which will stimulate apc present in or near the tumor , tumor - infiltrating dendritic cells . these dendritic cells may migrate to the dln where it is possible that it will stimulate a t - cell response to the tumor antigen . concluded that two mechanisms might have occurred to cause tumor regression : ( 1 ) a dth response to the tnp - modification of tumor cells , eliciting anti - tnp ctl , b cells , and dth responses in the tumor site or ( 2 ) the bystander effect of anti - tnp ctl by amplification of anti - tnp helper t - cell activity . neither of these mechanisms has been confirmed , but the extensive mechanisms of chs were not as clear in 1984 , so it is likely that the mechanisms are far more complicated than that , and that there are a slew of chs - effectors involved in the tumor regression . as highlighted before , there is no experimental or mechanistic explanation of a bystander effect , only observational . the mechanisms of contact hypersensitivity are hard to apply to this context , as the reactions are being induced in a tumor suppressive environment , which may not include many immune cell types . on top of this , the induction of hapten - mediated cell death must be considered , as it likely induces tumor regression and immune responses ( table 2 ) . it is very possible that the tumor regression is due to cell death of all the tumor cells or some combination of cell death and haptenation of the tumor cells . when speculating in this context , it is important to remember that tumor cell death in the tumor can elicit antitumor immune responses , although the type of cell death necessary to mediate immunity remains unclear . as seen in table 2 , it has been shown that in some systems , autophagy from chemotherapy induced the release of hmgb1 and atp , causing the recruitment and activation of dendritic cells and t - cells . the atp release may be similar to that seen in chs , where hapten modification causes atp release , stimulation of psrx7 on dendritic cells , and nlrp3 activation . this leads to il-18 and il-1 release , which can activate dendritic cells in the area . along with this , haptenation of the tumors may induce the upregulation of chs chemokines , selectins , and adhesion molecules in the tumor vasculature , causing recruitment of hapten - specific t and nk cells . [ 32 , 33 ] used a relatively high concentration of tncb in large injection volumes , so it is plausible that many of the cancer cells were going to be tnp - bound and died . low concentrations of haptens induce apoptosis , and higher concentrations , like used in fujiwara 's work , seem to cause necrosis [ 146 , 148 ] . hapten - mediated cell death must be considered as a viable mechanism for in situ haptenation - induced tumor regression . theoretically comparing hydrophobic and hydrophilic haptens , such as tncb and tnbs , respectively , could test this , where tncb kills bound - cells and tnbs allows further proliferation and growth of bound - cells . a tumor regression experiment using in situ haptenation injection with these two haptens ( separately ) in hapten - sensitized mice would show if it is the tnp haptenation leading to antitumor immune responses , the hapten - mediated cell death that is eliciting tumor regression , or some combination of both . the contact allergens for topical treatments of various dermatological problems , such as alopecia areata , viral warts , and some cutaneous tumors , have been used since the 1960s . buckley and vivier   reviewed many of the clinical trials using contact sensitizers to induce viral wart regression . they pointed out that very few of these studies had the proper control groups or randomization , making many of the observations biased and hard to gather conclusions from . the sensitizers mainly used for these trials were dncb , a potent contact allergen and mutagen first used in 1912 , squaric acid dibutyl ester ( sadbe ) , a potent contact allergen first used in 1979 , nonmutagenic , and commonly used to treat viral warts in europe and southeast asia , and diphencyprone ( dpcp ) , a potent contact allergen in humans and animals , nonmutagenic , and commercially available in the uk . all patients given this treatment were usually sensitized under the armpit with ~2% solutions of the hapten . the hapten was then applied to the warts at a concentration of 0.1% ( depending on location ) and was increased depending on the reaction seen . the mechanism of action for these contact allergens affecting viral warts is not well investigated , although it is theorized that the allergen application induces alterations in cytokine levels , nonspecific inflammation causing wart regression , and haptenation inducing hapten - specific immune responses . it is likely that chs / acd - like reactions are occurring in the wart site , although there is little evidence for this . it was seen that cd8 + t - cells infiltrate into warts upon dpcp application , and dncb application can increase complement - binding wart virus - specific antibodies . overall , the clearances of warts ranged from 7 to 100% in the trials with a median clearance rate of 62% . it was also seen that long - term , hapten - dependent treatment was needed to cause regression . upitis and krol   conducted a clinical trial using the hapten diphenylcyclopropenone ( dpc ) to treat recalcitrant palmoplantar and periungual warts . the study had 154 patients , all of which were sensitized to dpc ; 135 of which had complete clearance of warts with an average of 5 treatments over 6 months . there were very few side effects to the treatments , leading the authors to the conclusion that dpc should be considered as a first line treatment for warts . however , the mechanism of action is not well explained . a more recent clinical study , treated six facial wart patients , who were not responding to other treatments , with dpcp . patients were sensitized to 2% dpcp as described above , and various concentrations of dpcp were applied to the warts of interest in 810 sessions . four of six patients had complete disappearance of the warts with no recurrence for a year and the other two patients had improved warts . both of these studies seemed to be hapten - dependent phenomena . despite the evidence suggesting that contact allergen application can treat warts , warts are known to spontaneously regress and disappear . many of these studies were over one year , and very frequently , warts will spontaneously regress within a one- to two - year period . along with this , the mechanism of this viral wart regression remains largely unknown and needs further elucidation , although it is likely that a hapten - dependent chs - like immune response would have occurred , as most patients were sensitized to the hapten prior to use . epifocal hapten application at cutaneous tumor sites to elicit chs - like immune reactions and primary tumor regression is a long - established and appealing concept . edmund klein reviewed multiple clinical uses of epifocal hapten application for the treatment of cutaneous cancers . he assessed studies on cutaneous neoplasms , where treatment of epitheliomas using chemotherapy was compared to hapten - induced ( 2,3,5-triethyleneiminobenzoquinone ) [ teib ] and dncb cutaneous hypersensitivity reactions at the tumor site . these cutaneous hypersensitivity reactions at the tumor site resulted in the regression of superficial basal cell carcinomas ( bcc ) , squamous cell carcinomas ( scc ) in situ , and premalignant keratosis . in particular , multiple studies on patients with bcc where hypersensitivity was induced by topical application of cream containing 0.05% teib were described . a case study was done on one patient receiving this treatment , who had regression of several hundred basal cell carcinomas after 3 weeks of daily topical application . the tumors would become eurythmic , exudated , and necrotic within 24 hours of application . whenever the patient developed new lesions in different sites , the cream was applied and the tumors would disappear . there were also several studies performed on squamous cell carcinoma . the carcinomas in situ responded very well to topical challenge with teib or dncb and the reaction was similar to that seen in the basal cell carcinomas . more than 90% of the lesions underwent regression following the hapten challenge , although the deeper lesions responded poorly and did not fully regress , needing secondary treatment with the hapten , chemotherapy , or other standard treatment to eradicate it . these studies clearly demonstrate the powerful ability of haptens to cause chs reactions in epidermal tumor sites to cause local tumor regression . to note , the hapten - mediated tumor regression did not cause regression of untreated tumors suggesting that hapten - dependent tumor regression was mediated by cell death and/or chs - like reactions . truchetet et al .   reviewed the use of dncb in the treatment of metastatic melanoma in the clinical settings . most of these studies used epifocal dncb application at a concentration of 110% in acetone , some using sensitization and some not . in 1978 , loth and ehring   tried the treatment in 35 patients , nine of whom had a favorable response . in 1981 , picrard et al .   described 86 cases of primary melanoma with or without metastases treated with dncb after sensitization . all the patients benefitted from the epifocal applications of dncb on tumor and normal skin between the primary melanoma and excision of metastases . the 5-year survival was 77% with dncb application before and after resection versus 70% with dncb application only after resection . there was no survival benefit seen when the disease had spread to the lymph node . they state that dncb treatments are only useful for local recurrences and skin metastases , not surgically inaccessible regions . this would imply that the reaction is directly hapten - dependent and a bystander effect is not occurring in a majority of patients as the reactions may be limited to the skin lesions . the mechanism of tumor regression and whether it is mediated by hapten - cell death or chs like immune reactions was not studied . treated 59 recurrent melanoma patients with a combination of topical dncb and systemic dacarbazine ( dtic ) . patients were sensitized to 2% dncb on their cutaneous metastasis on day 1 and day 8 , followed by additional treatment on day 15 . dtic treatment was started 4 weeks after the first dncb application with 3 consecutive doses of 400  mg / m , a single dose of 800  mg / m , or 5 consecutive doses of 250  mg / m and repeated every 3 - 4 weeks . of the 59 patients , 15 ( 25% ) had a complete response , 7 ( 12% ) had partial response or stable disease , and 37 ( 65% ) had tumor progression . the presence of severe local reaction to topical dncb application correlated with improved overall survival . of the 15 complete responders , this study does not compare the data collected to dtic only treated patients , which is reported to have a 10.2% response rate in stage iv melanoma patients . dncb treatment only was also not studied , making it difficult to determine which treatment had an effect . however , they did state that no dncb - treated lesions disappeared until the start of dtic treatment . along with this , they sensitized patients at the tumor site , potentially diminishing the immune reactions as tumors are immune - suppressive . it would have made more impact if the hapten sensitization was given elsewhere as done in many other clinical settings using contact sensitizers to treat metastatic melanoma . although this study shows a few patients responding to the treatment , the data is not strong enough to suggest a positive response to the treatment . there have been many case studies using epifocal dncb or dpcp treatments for melanoma metastases [ 36 , 3840 ] . von nida and quirk   described a patient who was sensitized to 2% dncb on normal skin and once the appearance of low - grade eczema appeared at that site , the patient was instructed to apply 2% dncb to the tumor nodules . within 2 weeks this went on for 7 years until the patient had liver metastasis and succumbed to the disease . the dncb treatment in this case seemed to slow the progression of disease by treating cutaneous lesions in a hapten - dependent manner but did not ultimately stop the disease from metastasizing . damian et al .   described seven case studies of metastatic melanoma patients who were sensitized with 2 drops of 2% dpcp in acetone on the upper inner arm for 48 hours . two weeks after sensitization , dpcp aqueous cream was applied weekly to all cutaneous melanoma metastases . all of them had either slowing of tumor growth or regression of tumors where the dpcp was applied . three of the patients succumbed to the disease due to metastases within 5 weeks to 19 months , but four were alive at the time of publication . in a follow - up study , the role of th17 cells in one patient who remained free of cutaneous and regional disease for 4.5 years after dpcp and dtic treatment of the disease was reported . rna expression analysis revealed upregulation of the human th17 genes ( l-17a / b / c / d / e / f ; cd27 ; cd70 ; plzf-1 ; ctla-4 foxp3 and rort ) in the posttreatment tissue sections . this was not confirmed by looking at the presence of th17-associated protein or increased th17 cell infiltration . lastly , another group   reported a patient treated with the same method as damian et al . ,   which had regression of melanoma nodules on the ankle for up to 18 weeks . this area was dry and eczematous with the appearance of numerous eosinophils ( determined by h&e statin , no specific eosinophil markers ) and no melanoma ( hmb-45 stain ) . showing complete regression of merkel cell carcinoma in the scalp 1 year after treatment using a topical dncb treatment . the patient was sensitized to 2% dncb and dncb was applied to the lesions for 4 subsequent weeks . h&e immunostaining of biopsied specimens showed infiltration of cd3 + t - cells and cd28 + , kp-1 + macrophages . to note , mitoses of the tumor cells were still present , but much less frequent than before treatment . although these case studies [ 36 , 3840 ] suggest a beneficial aspect of the dncb or dpcp treatment , it is difficult to interpret these results , as case reports are typically the best - case scenario and are from rare patients that have a response . along with this , it is challenging to compare the study by strobbe et al .   and the case studies [ 36 , 3840 ] , as strobbe et al .   sensitized patients at the tumor site , which is immune - suppressive and may have dampened sensitization , while the case studies sensitized patients at distant skin sites , allowing for appropriate sensitization . something that all these studies do show is that the tumor regression seems to be hapten - dependent and seems to not induce a bystander effect , evident from metastases formation . there were very few immune correlations made in any of these studies , only visual observations , making it difficult to interpret how these treatments are inducing tumor regression . it would be interesting to expand the observations by klein   and perform a controlled trial in bcc or scc patients to establish if this method can indeed induce tumor regression , decrease recurrence of metastatic disease , and potentially increase the patient survival . 's   work utilizing dtic and dncb and examined the tumor regression mechanisms in b16f17 , slow growing b16 substrain , bearing c57bl/6 mice . seven days after subcutaneous tumor inoculation , when the tumor was 25  l in volume , mice were treated with i.p . injection of dtic and/or epifocal ( on the skin of the tumor site ) dncb application ( 25  l in acetone and olive oil , 4  :  1 ) 24 hours later . the concentration of both dtic and dncb was optimized to be 50  mg / kg dtic on days 7 , 12 , 16 , and 20 and 3% dncb on day 8 ( to mimic chs sensitization ) and 1% dncb on days 12 , 16 , and 20 . this treatment regimen resulted in tumor regression and tumor - free mice for up to 150 days in 72% of mice . lastly , whether or not this treatment would cause tumor regression or resistance of b16f17 lung metastases injected i.v . on day 7 dtic and dncb combination treated mice had significantly less lung metastases than the control and untreated groups 30 days after inoculation . interestingly , there was no single treatment controls used in many of these experiments , making it difficult to see the effect of the combined treatment compared to the individual treatment effects . ( 1 ) the animals were not sensitized to dncb using normal sensitization procedures . typically , for chs reactions , animals are sensitized to the hapten five days before challenge and are sensitized on the distant area ( usually the abdomen ) from the challenge . the effective sensitization time for dncb ( 5 days ) was not given and moreover , the sensitization was elicited on the tumor , which is immune suppressive . the authors mention that they tested the sensitization of different percentages of dncb using the ear - swelling test , but it is unclear if the dncb in this setting was applied on the tumor or in a different area of the animal . if the ear - swelling test was performed after sensitization at the tumor site , it would have been prudent to compare the ear swelling to mice sensitized at a nontumor site to see if the sensitization was affected by doing it at the tumor site . ( 2 ) all of the tumor measurements here are mean tumor volumes , yet there are no standard deviation or error bars on any of the points . it is difficult to tell what the range of data is and its relative significance . ( 3 ) appropriate controls were not used for each experiment ; dtic treatment alone or dncb treatment alone was given in the first figure and did not reflect in any subsequent figure . this makes the results difficult to interpret because it is unclear if it is the combination treatment or just a single treatment that caused the observed primary or pulmonary tumor regression . performed a follow - up study using this model to look into the antitumor immune responses elicited by the dtic / dncb combination treatment . they first repeated their previous results showing that 5 of 7 mice underwent complete tumor regression in the 35-day observation period . they looked at the incidence of pulmonary tumors after 7 treatments ( the last study used only 4 treatments ) and observed that there were only 7  4 tumors in the combination group versus 133  31 in control mice . splenocytes from treated animals that underwent primary tumor regression were tested for their ability to kill b16-melanoma cells in vitro using cr - release assay . the cytotoxicity of splenocytes from treated animals toward b16s was 3 times higher than control animals ; these splenocytes also released more ifn. macs isolated and in vitro restimulated cd4 + and cd8 + t - cells each from treated splenocytes had higher killing than the control , whereas the nk cells had similar killing as the control . the similar nk cell killing was expected , as nk cells involved in chs are derived from the liver   and not the spleen . ability of tils from the primary b16 tumor to kill b16 melanoma cells and release ifn in vitro was higher in the treated versus untreated animals . these cells also had high mrna levels of ifn , tnf , and il-6 . using rag mice , the paper also showed that tumor regression was dependent on t - cells and that this model was repeatable with another hapten , oxazolone . ( 1 ) as highlighted before , the single treatment controls were not looked at for any experiment , making it hard to tell if the ability of immune cells to kill or produce cytokines in vitro is from the combination of treatments or just one treatment alone . ( 2 ) for the cytotoxicity studies using cd4 + t - cells , cd8 + t - cells , and nk cells , they only stimulate these cells with melanoma in vitro , not stimulating the cells with dnp - modified melanoma to see if this has an ability to cause cytotoxicity . it is very likely that nk cells will not kill unhaptenated cells because of inhibitory molecules binding to mhc , as previously described . it is hard to draw conclusions from these cytotoxicity assays as stimulation with melanoma and dnp - bound melanoma was not compared . ( 3 ) the study used immune cells from the spleen , even though it is commonly known that chs - related t - cells mature and preside in the draining lymph nodes and chs - related nk cells reside in the liver . it is very possible that the collected cells had nothing to do with the treatment . despite the highlighted issues , these two papers establish the only mouse - model of tumor regression utilizing epifocal hapten application . however , these papers do not elucidate how the tumor regression is being mediated . to further elucidate the validity of this method , these experiments would need to be repeated with all the appropriate single treatment controls taking into consideration the extensive issues present in each paper . when considering the use of epifocal hapten application to induce chs - like immune reactions at the tumor site , two aspects must be taken into account : ( 1 ) haptens will induce cell death and chs - like immune reactions that may be able to cause tumor regression by utilizing the extensive immune cell milieu ( table 2 ) . ( 2 ) haptens will induce chs - like immune reactions that may lead to tumor cell growth and increased immune suppression ( table 3 ) . it is likely that epifocal hapten application induces tumor regression through chs - like mechanisms ( table 2 ) . first , epifocal hapten application would induce massive cell death in the tumor as any haptenated tumor cell would likely die . in a hapten presensitized animal , tumor haptenation and cell death will cause the release of danger signals , atp , and ros . atp release will induce p2rx7 , which will cause the activation of nlrp3 on apcs , eliciting the production of il-18 and il-1 ; these elicit protection against colorectal tumorigenesis by polarizing ifn+ release of ros has the ability to inhibit myeloid derived suppressor cell ( mdsc ) maturation , known to suppress immune responses against tumors by releasing il-10 , and induce cell death of tumor cells in the established tumor . the stimulation of apcs by danger signals could potentially reactivate exhausted cd8 + t - cells in the tumor microenvironment as dcs are linked to t - cell exhaustion [ 118 , 119 ] or help apcs traffic to the lymph node to establish new cd8 + effector t - cells . inkt - cells , activated by cd1d presentation of haptenated tumor glycolipids , and  t - cells will work together to produce ifn , which has an antitumor protective role as a potent th1 cytokine   and mediates antitumor activity . inkt - cell activation will also lead to il-4 release causing the activation of cs - initiating b-1 cells to produce hapten - tumor igm . this hapten - tumor igm will also lead to the activation of mast cells which will release tnf and cxcl2 , causing cause fasl+ , perforin+ neutrophil cell infiltration . these neutrophils may be able to kill the tumor cells in the first 24 hours [ 121 , 122 ] and provoke release of cxcl1 and cxcl2 from the surrounding tissue , helping t - cells traffic to the tumor site . the mast cells will also release tnf and serotonin , causing upregulation of chemokines , selectins , and adhesion molecules and subsequent hapten - specific t - cell to trafficking to the tumor . hapten - specific cd8 + t - cells will enter the area and produce ifn , which can help to stimulate other effector tils in the area   and cause antitumor activity . along with this , the entry of hapten - specific cd4 + t - cells could potentially rescue exhausted cd8 + t - cells , as seen in chronic viral infections . the entry of tc17 and th17 cells could elicit multiple antitumor immune responses , as cd4 + and cd8 + il-17 producing t - cells have been shown to elicit tumor regression in melanoma mouse models [ 126 , 127 ] . lastly , hapten application could induce the infiltration of cxcr6 + hepatic nk cells , which may be able to cause tumor cell death once in the site . despite all the possible reactions that could occur , it is difficult to say if and how these responses would lead to a bystander tumor effect , as there is little evidence for the functionality of hapten - effector cross - reactivity . the only process that could lead to a bystander effect is the massive amount of cell death that occurs from haptenation , causing the release of tumor antigens into the animal and potential immune recognition against these antigens . there are many aspects of chs - like reactions that could cause tumor immune suppression and promote tumor cell growth , instead of regression . bock et al .   looked at the ability of continuous dnfb exposure to cause toxicity and tumor formation in multiple different mouse strains . in this study , the animals were exposed to one dose of 7,12-dimethylbenz[]anthracene ( dmba ) , a known cancer causing agent , and then applied 0.1%  dnfb to the site 5 times a week for 1450 weeks starting 21 days after the dmba . this caused 35/50 swiss , 6/30 c57bl/6 , and 5/30 balb / c mice to form tumors . dmba treatment alone resulted in very low incidence of tumors , 2/50 swiss , and 0/30 c57bl/6 and balb / c mice , respectively . there were no tumor formations in swiss mice ( 0/50 ) that were treated with only dnfb . the data suggest that although dnfb is not a causative agent of cancers , it is a tumor - promoting agent and can possibly cause tumor formation in predisposed conditions or already growing tumors with repeated exposure . it is important to note that massive amounts of dnfb were given to these animals over very long periods of time and the mechanism of hapten - mediated tumor promotion was not discussed . an extensive 24-year study , between 1984 and 2008 , by engkilde et al .   looked at the association between contact allergy by small chemicals and cancer incidence . the group patch tested , a way of identifying whether a small molecule causes skin inflammation upon contact , 16,922 patients ( 6,113 men and 10,809 women ) , 35.8% of which had a positive reaction to at least one allergen . these results were linked to the danish cancer registry , where the group saw that 3,200 ( 18.9% ) of the dermatitis patients had some type of cancer and that 1,207 ( 37.7% ) of these patients had a positive patch test . the group found significant correlations between contact allergy and bladder , breast , and skin ( nonmelanoma ) cancer regardless of sex . there was also an inverse correlation between a positive patch test and brain / cns cancer in women . this study underscores that the reactions causing acd , like those involved in chs , may be associated with cancer in certain cases . we have conceptualized some of the possible mechanisms of hapten - induced chs promoting tumor immune suppression and tumor growth ( table 3 ) . epifocal application of a hapten will cause the release of danger signals , such as pge2 , ros , and atp . pge2 release has been seen to induce colon cancer growth   and cause mdsc activation in the tumor site . ros release is known to upregulate vegf , promoting angiogenesis in tumor sites , and possibly cause the nitration of t - cell - peptide - mhc interactions , inducing t - cell suppression . atp release will induce p2rx7 , which will cause the activation of nlrp3 on dermal apcs , eliciting the production of il-1 and il-18 which has been shown to decrease the tumor responsiveness to certain vaccinations . the danger signal release will cause tlr4 and tlr2 stimulation of dermal apcs , which has been shown to elicit immune evasion by helping myeloid cells establish metastases via tgf- [ 115 , 116 ] . haptenation will also cause keratinocytes to release il-1 , il-6 , il-18 , and tnf , which have been shown to cause mdsc recruitment and infiltration at the tumor site , subsequently causing il-10 release and immune suppression . inkt - cell activation will cause release of il-4 and il-13 , which are both known to elicit mdsc recruitment and infiltration   as well as direct suppression of tumor - specific cd8 + t - cells . mast cell activation by complement c5a will cause ccl2 and ccl5 upregulation , which has been to shown to induce tumor associated macrophages ( tams ) to release il-10 , promote angiogenesis , and stimulate tumor metastasis . mast cells will also release tnf , known to help deliver oxygen to hypoxic areas of the tumor allowing for tumor growth , and release cxcl2 , seen to induce melanoma cell proliferation . lastly , the induction of chs at the tumor site could cause the infiltration of hapten - specific t - regs , which could potentially release il-10 to suppress effector t - cells   or elicit cd8 + t - cell exhaustion by expression of ctla-4 . it is likely that the antitumor immunity or tumor - mediated immune suppression and tumor growth due to elicitation of chs from epifocal hapten application will have much to do with the ( a ) type of tumor treated ( b ) growth rate of the tumor , and ( c ) timing of the administration . it is suggested , by hapten - specific t - cell migration data , that no antigen presentation occurs outside of the dermis in the chs elicitation phase . this finding makes it likely that epifocal hapten application will only be useful for treating cutaneous cancer . the mechanisms of hapten - induced tumor regression using epifocal hapten application still remain unclear and need to be further studied . it is also essential to figure out the situations in which a hapten will induce tumor regression versus tumor growth by testing several different haptens in well - defined systems , which have yet to be created . if all this is done , it can be understood if epifocal hapten application is useful in eliciting tumor regression and antitumor immune responses . from 2002 to 2013 , philip s. low 's group used a unique approach to hapten - mediated tumor treatment . they synthesized folate - hapten conjugates and used them to treat folate receptor high cancers . the concept is that the folate would bind to folate receptors on the tumors coating the tumors in haptens , which could lead to adcc and complement system activation , effectively killing the tumor in hapten - sensitized animals . in their work , they utilized the haptens fitc and dnp , and treated folate high m109 lung carcinomas . it is important to note that the immune mechanisms occurring here are wildly different than what has been described earlier ( sections  1 , 2 , and 3 of the hapten - mediated tumor treatments ) having little to do with chs mechanisms , and mostly mediated by hapten - induced adcc . they treated cancer cells in vitro with the folate - fitc conjugates , ensuring the fitc coating of m109 cells . balb / c mice were inoculated with m109 cells and sensitized to bsa - fitc , inducing a strong anti - fitc antibody response . they observed slight increase in survival in mice with peritoneal m109 tumors with the il-2 or folate - fitc alone ( i.p . they added ifn treatment to the il-2 + folate - fitc , which showed a very significant increase in survival , from a maximum of 30 days up to over 80 days in 20% of the animals . after immense optimization of folate - fitc , il-2 , and ifn concentrations , they were able to find a curative treatment that gave 100% survival of mice for 100 days . they rechallenged long - term survivors with the same number followed by 3x as many m109 cells and saw that the mice were able to survive the rechallenges , suggesting long - term immunity in these mice ; this was only shown as survival curves , so it was unclear if the tumors grew or not . of note , many cells in the body express the folate receptor and this treatment could cause fitc coating and adcc at distant , folate receptor expressing sites . realizing this , the authors submitted cured animals for toxicological analysis where it was determined that the treatment was not toxic and that there was no opsonization or damage of organs . along with that , il-2 and ifn treatments are known to cause side effects in clinical use , so combining them with the folate - fitc conjugate could increase any potential side effects . despite these worries , they clearly showed that this method coated tumors cells in vivo with fitc and significantly increased mouse survival in combination with cytokine treatment . lu et al . ,   then studied the immune mechanisms of folate - fitc - mediated tumor regression . they observed a bimodal plot of folate - fitc at various concentrations ; this is commonly seen in treatments that do not directly kill tumor cell . there was no complement - mediated lysis of folate - fitc - labeled tumor cells occurring . nk cells showed direct lysis of folate - fitc coated tumor cells in the presence of anti - fitc antibody , suggesting adcc . macrophages engulfed the folate - fitc - bound tumor cells opsonized with fitc antiserum and ~34% of these cells were engulfed after a 30-minute coculture . these data suggest that both nk cells and macrophages are involved in killing and clearing folate - fitc / anti - fitc antibody marked tumor cells . using the complete treatment in vivo , they compared the survival of treated control mice and nk cell - depleted mice , showing a decrease in the overall survival , back to the basal level without nk cells . depletion of cd4 + t - cells and cd8 + t - cells alone and in combination and depletion of macrophages significantly decreased the overall survival of the mice , close to that of the untreated mice , but not as extreme a decrease as the nk cell depletion . cd8 + t - cells were removed from cured animals and were seen to kill m109 cells better than t - cells from untreated animals , suggesting that this treatment is eliciting t - cell memory against the tumor . however , they did not perform adoptive transfer experiments to see if these cells could clear m109 tumors in nave animals . lastly , they showed that the optimized treatment was able to fully regress the tumor for 35 days , whereas the controls ( pbs and pbs + il-2/ifn ) had little effect . these papers provide strong evidence for folate - fitc - mediated tumor regression and underlying immune mechanisms of this regression . however , it must be determined what the role of cd4 + and cd8 + t - cells is in this treatment and how the animals are clearing secondary tumor challenges . it is likely that macrophages are presenting tumor antigens after opsonization , causing the formation of tumor - specific t - cells . this is likely the reason cd4 + and cd8 + t - cells are important for animal survival . they utilized a radioactive folate - fitc conjugate to track the movement of the conjugate in vivo and saw that it was rapidly eliminated in nave mice but formed immune complexes with fitc - specific antibodies in fitc sensitized animals , causing an extended duration of folate - fitc in the animal ( 173-fold increase in drug exposure ) . extremely high doses of the folate - fitc were shown to cosaturate the tumor cell 's folate receptors and the circulating fitc - specific antibodies , hindering immune recognition of the tumor and thereby lowering the antitumor activity . also established folate - dnp conjugates ( ec57 , ec63 , ec0293 , and ec0294 ) that showed similar results to the folate - fitc conjugate when using similar treatment regimens . one ( ec0294 ) of four tested - conjugates , in combination with il-2 and ifn , markedly improved survival of m109 tumor bearing mice for more than 100 days ; two of the treatments , ec0293 and ec0294 , gave 40 and 60% cure rates , respectively , among these mice . the cured mice all rejected the secondary tumor inoculation of m109 cells , suggesting an antitumor immune response . they looked into the risk of an allergic response , passive cutaneous anaphylaxis assay , to the treatment and saw that the conjugates that gave allergic responses were the ones that cured mice . these results show that the folate - dnp conjugates can elicit prolonged survival , secondary tumor rejection , and autoimmune side effects ; however , they do not show direct tumor regression results . this study shows that the concept of antigen - hapten treatment is a very effective treatment for folate receptor high cancers as it can be done with different haptens ( fitc and dnp ) and potentially elicits long - term tumor immunity . it would be interesting to know if other antigen - receptor targets could elicit similar results . recently , low 's group   published a phase i clinical study using the folate - fitc treatment alone in patients with renal cell carcinoma . patients were given ec90 , the hapten fluorescein , with the adjuvant gpi-0100 to stimulate the production of anti - fitc antibodies followed by ec17 , the folate - fitc conjugate treatment . 39 patients got at least one dose of the ec90 , and 33 received at least one dose of the ec17 treatment . of the 33 patients that received the ec17 treatment , 28 patients had baseline and at least one had follow - up tumor assessment . of 28 patients , 1 ( 4% ) patient achieved partial response , 15 ( 54% ) patients achieved stable disease , and 12 ( 43% ) had progressive disease . of the 16 patients that completed 2 cycles of the ec17 therapy , 12 ( 75% ) had stable disease and 4 ( 25% ) had progressive disease and of the 11 patients that completed 3 cycles of the therapy , 6 ( 55% ) had stable disease and 5 ( 45% ) had progressive disease . there was no apparent relationship found between the anti - fitc antibody titer and the best response to the therapy . although many patients had stable disease , only one had partial regression and no patients had complete regression . these results are not unexpected , as the mouse treatment required the use of il-2 and ifn treatments to be fully effective . in the clinical study , patients were also not sensitized to the hapten , likely affecting the results . this trial was likely performed to see the side effects of the folate - fitc conjugate alone on patients . as stated in the phase i study , low 's group has completed a phase ii trial of the ec17 treatment in combination with cytokine treatment and we hope those results will be published soon . it still needs to be determined how tumor challenges are rejected using this method . evidently , the field of contact hypersensitivity is still expanding , as there are many conflicting reports on several different aspects of the mechanism . the use of different mouse strains , different haptens , and different administrations or concentrations of haptens greatly impacts the immune responses seen . it would be paramount to attempt to standardize the methods of inducing chs , so that more clear mechanisms can be established between different haptens and mouse strains . there is much work to be done to fill in the gaps and confirm parts of the pathway that remain unclear . obviously , the use of haptens and haptenation as a tumor treatment needs further research to determine its efficacy . much of the work with hapten - inducing tumor regression was done before the field of chs was developed to its present state , and without in - depth immunologic mechanism depiction . this leaves much speculation about all the results found , as we underscored in this review . of the four concepts , antigen - hapten delivery seems to be the most appealing , but it uses completely different tumor clearance than the other treatment mechanisms , as it is mediated by adcc . the work done by low 's group [ 4347 ] is detailed in explaining the tumor regression mechanism ; however , further research is needed to understand if the treatment of folate - fitc along with il-2 and ifn can be effective . along with this , it must be understood how tumor rechallenges are rejected after treatment . for the field of hapten - mediated tumor regression to move forward , we propose that each model of hapten - mediated tumor regression be fully studied so that the mechanisms of primary and secondary tumor regression become clear . in this regard , we urge that the field must also consider the effect of hapten - mediated cell death , as the dead cells , like irradiated cells , may elicit antitumor immunity ; it needs to be determined if hapten modification alone ( on the surface ) or hapten modification followed by cell death is needed to mediate antitumor immune responses . it also must be determined whether or not hapten - induced tumor regression can induce bystander effects or if it is hapten - dependent . lastly , it is very important to realize that no hapten treatment has been effective without the combination of another immune- or tumor - modulating agent(s ) , suggesting that haptens may never be able to elicit complete tumor regression by themselves . if this is true , haptens may be considered as adjuvants to possibly increase tumor regression and antitumor immunity by combining them with other tumor treatments that have measurable efficacy . much of the data on hapten - mediated tumor treatments is observational ; thus more mechanistic studies using similar mouse models and haptens as well as more stringently - controlled clinical trials are essential to determine if haptens are appropriate as cancer immunotherapies .
haptens are small molecule irritants that bind to proteins and elicit an immune response . 
 haptens have been commonly used to study allergic contact dermatitis ( acd ) using animal contact hypersensitivity ( chs ) models . 
 however , extensive research into contact hypersensitivity has offered a confusing and intriguing mechanism of allergic reactions occurring in the skin . 
 the abilities of haptens to induce such reactions have been frequently utilized to study the mechanisms of inflammatory bowel disease ( ibd ) to induce autoimmune - like responses such as autoimmune hemolytic anemia and to elicit viral wart and tumor regression . 
 hapten - induced tumor regression has been studied since the mid-1900s and relies on four major concepts : ( 1 ) ex vivo haptenation , ( 2 ) in situ haptenation , ( 3 ) epifocal hapten application , and ( 4 ) antigen - hapten conjugate injection . 
 each of these approaches elicits unique responses in mice and humans . 
 the present review attempts to provide a critical appraisal of the hapten - mediated tumor treatments and offers insights for future development of the field .