Published in Volume 110, Issue = 3
J.=20 Clin. Invest. 110(3): 289-294 (2002).=20 doi:10.1172/JCI16216.
Copyright =C2=A92002 by the = American Society=20 for Clinical Investigation

Perspective

Cancer vaccines: progress reveals = new=20 complexities

Zhiya Yu and Nicholas = P.=20 Restifo

National Cancer Institute, National = Institutes=20 of Health, Bethesda, Maryland, USA

A decade ago, it seemed clear that our burgeoning = knowledge of=20 the molecular identities of tumor-associated antigens and a = deeper=20 understanding of basic immunology would point the way to an=20 effective therapeutic cancer vaccine. Significant progress = has been=20 made and objective regressions after immune-based treatments = are=20 observed in some patients =E2=80=94 even in those with = bulky, metastatic=20 disease. Notwithstanding this progress, we do not yet have a = cancer=20 vaccine in hand that can reliably increase patient survival = or=20 induce tumor destruction.

The creation of therapeutic cancer vaccines has proven to = be an=20 enormous challenge, and many of the strategies learned in = the=20 development of highly successful vaccines against infectious = agents=20 simply do not apply to cancer vaccines. Issues of antigenic = change=20 and immune escape are present in both antitumor and = antiviral=20 situations. However, one big difference between antiviral = and=20 antitumor vaccines is that the former are preventative = whereas the=20 latter are generally expected to be therapeutic.

Another problem with the targeting of tumor antigens = relates to=20 their poor immunogenicity. Tumor antigens appear to be = relatively=20 well tolerated in the host, perhaps because many of these = antigens=20 are also expressed in normal tissues. In this context, a = successful=20 cancer vaccine raises the specter of autoimmune attack if = the=20 vaccines are ultimately powerful enough to eliminate cancer = cells.=20 In this review, we highlight new challenges that have been = revealed=20 by recent progress in the field of tumor immunology. We then = attempt=20 to outline a future plan for cancer immunotherapy.

The existence of tumor-specific immune = responses=20 in cancer patients

The first reports that immune responses might result in = tumor=20 regression came over a century ago from William Coley, who = treated=20 cancer patients with live bacterial cultures, = nonspecifically=20 activating their immune systems. In the 1980s, Rosenberg et = al.=20 pioneered the use of high doses of the T cell growth factor = IL-2 in=20 individuals with metastatic kidney cancer or melanoma and = achieved=20 objective cancer regressions in 15=E2=80=9320% of treated = patients (1).=20 Because IL-2 is not known to have direct effects on the = growth of=20 solid tumors, its antitumor activities are most likely = associated=20 with its ability to expand lymphocytes, including the = low-affinity T=20 cells.

The most striking evidence for naturally occurring = antitumor=20 immune responses comes from rare anecdotal observations of=20 spontaneous regressions of tumors in patients with cancer. = Also=20 rare, but somewhat more amenable to study, is the = observation of=20 paraneoplastic autoimmunity that can accompany often occult=20 malignancies. For example, high titers of serum IgG specific = for=20 immunogenic proteins expressed in both carcinomas and normal = tissues=20 have been detected in cancer patients with paraneoplastic=20 neurological diseases (2).=20 Because these antigens are generally found in the nucleus = and=20 cytoplasm, it is unlikely that the autoantibodies have = direct=20 effects on either tumors or normal tissues. However, they = are=20 proposed to be surrogate markers for activated cellular = immune=20 responses. Indeed, cytotoxic T lymphocytes specific for = cdr-2 have=20 been detected and linked to cerebellar degeneration in those = seropositive patients with breast and ovarian cancers (2).

Autoimmunity has been modeled to a limited extent in = animals,=20 where vitiligo, the patchy and permanent depigmentation that = results=20 from the destruction of dermal melanocytes, has been found = to=20 accompany the regression of the experimental B16 melanoma in = C57BL/6=20 mice. Vitiligo is also positively correlated with a = favorable=20 response to IL-2 in patients with metastatic melanoma (3, 4).

Identification of tumor-associated = antigens=20 suitable for therapeutic targeting

A variety of techniques have been applied to identify = tumor=20 antigens recognizable by tumor-specific T cells. None has = been more=20 successful than an approach that uses transient transfection = of=20 pools of genes from a tumor-derived cDNA library to confer=20 recognition upon a target cell, thus identifying the gene = encoding=20 the target epitope. While cloning efforts have been = prodigiously=20 successful, protein chemists have also made inroads into the = identification of target antigens by pushing the limits of=20 high-performance liquid chromatography and tandem mass = spectrometry.=20 Peptides can now be eluted from MHC complexes derived from = tumor=20 cell membranes and characterized directly (5). In=20 addition, it is possible to test candidate tumor antigens by = the=20 so-called reverse immunology method, specifically by = sensitizing=20 immune cells with the candidate antigen, then testing the = ability of=20 sensitized cells to specifically kill tumor cells that are = known to=20 express the antigen.

To date, approximately 70 MHC class I=E2=80=93 and class = II=E2=80=93associated=20 tumor antigens have been discovered, while more than 1,700 = have been=20 identified by antibodies in cancer patients. About ten = antigens are=20 currently known to be recognizable by both T cells and = antibodies,=20 although the actual number of antigens for which IgG = production=20 requires Th cells is probably much greater.

We do not know for certain how many of the candidate = tumor=20 antigens are suitable targets for tumor immunotherapy. A = =E2=80=9Cvalid=E2=80=9D=20 target antigen must be expressed specifically in the tumor, = or at=20 least be expressed at levels sufficiently higher there than = in vital=20 organs. For T cell=E2=80=93based therapy, it must be = processed and presented=20 in the context of MHC molecules. The need for positive and = negative=20 control tumor lines is often overlooked; rigorous (and = numerous)=20 controls are needed to convincingly demonstrate that a = candidate=20 antigen is a suitable target for use in an immunotherapy = trial.

It has been estimated that 10⁵ to = 10⁶=20 MHC/peptide complexes are present on the surface of a = typical cell=20 (6),=20 although some antigen-presenting cells may express more. = Considering=20 the redundancy of individual complexes in a given cell and = the poor=20 MHC expression and antigen processing in the majority of = tumor=20 cells, a maximum of approximately 10,000 different = MHC/peptide=20 complexes is likely to be presented on a tumor cell. With = the=20 assumption that dozens of peptide epitopes could be derived = from an=20 expressed protein, and additional antigens could be = generated from=20 alternative open reading frames, a conservative estimate of = the=20 total number of peptides that are actually able to bind any = given=20 MHC may exceed 1 million. Therefore, the chances that any = given=20 peptide will be presented on the tumor cell surface are=20 approximately 1:100, which leaves 99% of the potentially=20 recognizable T cell epitopes simply absent from the surface = of a=20 given target tumor cell. Therefore, it is clearly not valid = to=20 assume that expression of a mutated candidate antigen will = result in=20 the MHC-restricted presentation of that antigen on the tumor = cell=20 surface.

Despite practical and theoretical concerns about some = putative=20 tumor-associated antigens, there is already a large and = growing list=20 of antigens that have been convincingly shown to be valid = targets=20 for immunotherapy. The success of tumor antigen = identification=20 approaches put to rest the notion that spontaneous human = tumors =E2=80=94=20 unlike their experimental mouse counterparts =E2=80=94 = simply lacked the=20 expression of antigens recognizable by the immune = system.

Enhancing tumor antigen immunogenicity = by=20 modifying epitope sequences

Tumor antigens in their original form generally bind = poorly to=20 their restricting MHC molecules. In addition, peripheral=20 autoreactive T cell precursors recognize their cognate = peptide/MHC=20 complexes with low affinity. Thus, most tumor antigens = identified so=20 far are poorly immunogenic in vivo. Dramatic increases in = the=20 magnitude of T cell responses and sensitivity to antigen = stimulation=20 have been observed in both human and mouse models using = agonistic=20 altered peptide ligands. These altered peptides are capable = of=20 enhancing the stability of peptide/MHC complexes because of=20 modifications in the MHC anchor residues (7) or as=20 the result of favorable and generally conservative changes = to the=20 peptide at the interface with the T cell receptor (TCR) = (8, 9). Most=20 importantly, the enhanced T cell responses can retain their=20 specificity to the native antigen, which allows them to kill = target=20 tumor cells ex vivo and, presumably, in vivo.

Both the association and the dissociation rates = contribute to the=20 steady-state stability of the interactions between = peptide/MHC and=20 ultimately TCR/peptide/MHC complexes. However, we have found = that=20 the enhancement of immunogenicity might be more affected by = the=20 off-rate than by the on-rate. For example, = gp100_209(2M),=20 an anchor-fixing modification of a native antigenic peptide = derived=20 from human melanoma antigen, gp100_209-217, is = 100-fold=20 more potent in activation of naive T cells than is wild-type = peptide. The steady-state binding affinity of the modified = peptide=20 is nine times higher than that of the wild-type peptide. In=20 contrast, the dissociation rate of modified peptide from = HLA-A2=20 molecule is more than 100-fold slower than that measured = with the=20 wild-type peptide (our unpublished data).

The stability of the target peptide/MHC complex is not = only=20 important in order to achieve the required antigenic density = for=20 naive T cell activation but may also alter the quality of = the T cell=20 response. A study by Mark Davis=E2=80=99s group concluded = that a TCR bound=20 to antagonist ligands with lower affinities because of an = increased=20 off-rate (10).=20 Conversely, extending the off-rate by amino acid = substitutions may=20 augment T cell =E2=80=9Caffinity maturation.=E2=80=9D = Immunizing mice with agonistic=20 peptide ligands elicits high-avidity T cells that can = recognize the=20 relevant tumor cells, and even target cells pulsed at = relatively low=20 concentrations with weakly binding peptides (8).=20 Studies from our laboratory have shown that although the = human=20 (hgp100_25-33) and mouse (mgp100_25-33) = epitopes=20 are homologous, differences in the three = NH₂-terminal=20 amino acids result in a 2-log increase in the ability of the = human=20 peptide to stabilize =E2=80=9Cempty=E2=80=9D D^b = MHC molecules and a 3-log=20 increase in its ability to trigger IFN-=CE=B3 release by T = cells (11). In=20 a clinical trial, using anchor-modified gp100 peptide = immunization=20 in melanoma patients resulted in dramatic increase of = tumor-reactive=20 T cell responses (12).=20 Thus, modified antigenic peptide based on these ideas could = be of=20 significant value in vaccination against tolerant or weakly=20 immunogenic tumor-associated cells.

The nature of antitumor effector=20 cells

More than 40 years ago, Prehn and Main obtained evidence = of=20 specificity in the immune response to tumors. Mice immunized = with=20 irradiated methylcholanthrene-induced sarcoma cells, they = showed,=20 are fully protected against a subsequent challenge with that = same=20 tumor, but not with other tumors. Humoral responses against = solid=20 tumors may play some role in the effective killing of tumor = targets=20 either by augmenting antigen presentation or by the ligation = of a=20 growth factor receptor on cancer cells, such as HER-2/Neu. A = large=20 body of work using antibody depletion and gene knockout mice = has=20 revealed that both CD8⁺ and CD4⁺ T = lymphocytes=20 are crucial for therapeutic antitumor immune responses.

Compared with the comprehensive studies using = CD8⁺ T=20 cells in tumor models, relatively little is known about how=20 CD4⁺ T cells influence antitumor immunity. Very = early=20 work demonstrated that disseminated murine leukemia could be = eradicated by a combination of cyclophosphamide and = adoptively=20 transferred cells, now known to be CD4⁺ T cells = (13).=20 The most dramatic examples of the power of CD4⁺ T = cells=20 in the immune response to self-proteins can be found in = murine=20 models of autoimmune diseases, such as experimental allergic = encephalomyelitis, systemic lupus erythematosus, and = diabetes. In=20 these models, disease can often be transferred to naive mice = with=20 purified, self-reactive CD4⁺ splenocytes or = specific=20 CD4⁺ T lymphocyte clones. Antigen-specific=20 CD4⁺ T lymphocyte clones can also treat tumor = through=20 CD8⁺ T cells specific for the cognate antigen = (14).=20 Tumor antigen=E2=80=93specific CD4⁺ T cells have = been isolated=20 from tumor-infiltrating lymphocytes from melanoma patients = (15).=20 Adoptive transfer into patients of unfractionated = tumor-specific T=20 cells has been shown to promote tumor regression (16).=20 These studies and others suggest that the full activation of = autoreactive CD4⁺ T cells may be an important = immune=20 component that is currently missing from many current = clinical=20 cancer vaccine trials.

Natural killer cells kill many tumor cell lines and may = also play=20 a critical role in antitumor immunity. A recent study in = transgenic=20 mice lacking NK1.1⁺CD3^=E2=80=93 cells = linked an=20 impaired acute tumor rejection to deficiency of NK activity = (17).=20 NKG2D receptors expressed by NK cells and activated = CD8⁺=20 T cells and macrophages can be stimulated by their ligands, = which=20 are often overexpressed on cancer cell lines (18).=20 Most surprisingly, clinical results from hematopoietic cell=20 transplantation revealed that alloreactive NK cells in the = donor=20 graft prevented leukemia relapse in leukemic recipients = (19).

Although the innate immune system is often ignored as an=20 important component of the antitumor immune response, = lessons=20 learned about immunity to infectious microorganisms indicate = that=20 early host defenses help determine the nature of downstream = adaptive=20 immune responses. One recent line of investigation pursued = in our=20 laboratory concerns the initiation of apoptotic death that = results=20 in the engagement of key innate immune pathways; the = consequences of=20 cell death may induce dendritic cell activation and benefit = immune=20 induction (ref. 20; see=20 also Steinman and Pope, this Perspective series, ref. 21). A=20 new generation of nucleic acid vaccines encoding an = alphaviral=20 replicase enzyme, together with tumor antigens, induces = apoptotic=20 death coupled with antigen production. This approach leads = to=20 quantitative and qualitative enhancement of the therapeutic=20 antitumor immune response in animal models (22,=20 23).

Unexpected obstacles in early clinical = trials

With antigens in hand, immunotherapists set off to create = a new=20 class of therapeutic vaccines based on defined antigens. = Recombinant=20 immunogens were created using the same viruses that have = proven=20 themselves to be so successful in the realm of infectious = diseases =E2=80=94=20 including vaccinia, polio, and influenza A =E2=80=94 as well = as some others=20 including adenoviruses and bird poxviruses (canarypox and = fowlpox)=20 (24,=20 25). In=20 animal models, these vaccines can prime T cell responses and = elicit=20 powerful immune responses that lead to tumor cell = destruction.=20 However, when these viruses were tried in the clinic, it = became=20 apparent that experiments in animal models had failed to = predict key=20 aspects of recombinant vaccine function in people.

One reason for these disappointing results was = pre-existing=20 neutralizing antibody. In one recent study in which patients = were=20 immunized with recombinant adenoviruses encoding the=20 melanoma-associated antigens gp100 or MART-1, only 6 of 54 = patients=20 had neutralizing antibody titers of less than 100, with the = majority=20 having neutralizing titers of more than 400 (26).=20 Similar problems might be observed using clinical-grade = recombinant=20 vaccinia viruses. Patients thus retain strong anti-vaccinia=20 antibodies for many decades after immunization =E2=80=94 a = lasting legacy of=20 the worldwide immunization program to eradicate smallpox. = One would=20 fully expect pre-existing immunity to be a problem in the = use of=20 several other vectors under consideration, including = recombinant=20 versions of polio and transfectant influenza A viruses of = the more=20 commonly observed strains. One way to circumvent this = problem would=20 be to use viruses whose natural hosts are non-mammalian, = such as the=20 avian poxviruses (Letvin, this Perspective series, ref. = 27).=20 These viruses are antigenically distinct from poxviruses and = are=20 incapable of replicating in mammalian cells. Strategies = employing=20 genetically engineered influenza viruses may prove useful in = the=20 development of live virus vaccines against cancer (24).

Another potential obstacle uncovered in early clinical = trials is=20 immunity to an antigenically complex vaccine that is = immunodominant=20 over a response to a transgene-encoded weak tumor antigen. = This=20 problem, which was not adequately studied in early = experimental=20 animal models and remains exceedingly difficult to model in = ongoing=20 preclinical work, may be summarized as follows: Vectors may=20 interfere with the induction of reactivity to the encoded = tumor=20 antigen through the poorly understood mechanisms of = immunodominance.=20 T cell responses elicited by protein immunization tend to = focus on=20 one or a few sites in the antigen. Whether this phenomenon = is driven=20 by the predetermined TCR repertoire, the competition among T = cells=20 based on their affinity to antigenic determinants, or the=20 characteristics of antigen processing is not known (28,=20 29). In=20 viral vector=E2=80=93based vaccines, self-antigens are = coexpressed with=20 viral proteins. If the immunodominant sites reside in the = viral=20 components, the vaccine will fail to elicit the desired = immune=20 responses. The use of vaccines based on = =E2=80=9Cnaked=E2=80=9D plasmid DNA vaccines=20 (i.e., DNA without associated protein) may circumvent both=20 pre-existing immunity and immunodominance. Despite these = advantages,=20 our own clinical work has shown no evidence of immunization = or=20 antitumor effect of naked DNA immunization against the gp100 = tumor=20 antigen (unpublished data), although naked DNA is effective = in many=20 animal models.

Consistent increases in tumor-specific = T cells=20 without consistent clinical responses

There is now incontrovertible evidence that precursor = frequencies=20 of tumor-specific T cells can be increased after = immunization using=20 several different tumor-associated antigens =E2=80=94 = including those=20 antigens that are nonmutated =E2=80=9Cself=E2=80=9D tissue = differentiation antigens=20 (12).=20 The presence of increased antitumor T cell precursors after=20 vaccination has been convincingly demonstrated in both mice = and=20 humans, using tetramer or ELISPOT analysis, real-time = RT-PCR, and=20 other techniques.

Thus far, the most effective immunization strategy in our = patients with advanced melanoma has been vaccination with = peptide=20 emulsified in incomplete Freund=E2=80=99s adjuvant. = Immunization with a=20 gp100-derived peptide modified to enhance its binding to = HLA-A2=20 dramatically increased levels of peptide-specific = CD8⁺ T=20 cells in the peripheral blood. Importantly, these T cells = recognized=20 and killed a variety of melanoma cells that expressed the = gp100=20 melanoma antigen and the restriction element HLA-A*0201 = after=20 culture ex vivo. Administration of IL-2 following peptide=20 immunization resulted in significantly more objective tumor=20 regressions than seen after IL-2 treatment alone (12).=20 However, most of these responses turned out to be partial = and=20 transient, and most responding patients eventually succumbed = to=20 progressively growing tumor.

Proposed mechanisms of tumor=20 escape

The current notion that tumor cells must = =E2=80=9Cescape=E2=80=9D immune=20 recognition is based largely on the idea that neoantigens = expressed=20 by tumor cells as a consequence of their genetic instability = will be=20 immunogenic. There is little doubt that the tumor contains a = large=20 number of mutations (30)=20 that can potentially generate new antigens recognizable by = the=20 immune system, but there is considerable doubt about what = the=20 immunological response to these potential immunogens will = be. A=20 number of groups have conducted experiments in which highly=20 immunogenic foreign antigens, such as the hemagglutinin = protein from=20 influenza (31),=20 the =CE=B2-galactosidase enzyme from Escherichia coli = (32),=20 and the ovalbumin protein from the chicken, are expressed in = tumor=20 cells (33).=20 The results are fairly uniform: tumors tend to grow = progressively,=20 retaining their lethality despite the expression of a = foreign and=20 highly immunogenic protein by the tumor cell.

Proposed mechanisms for tumor escape include those = relating to=20 the inherent genetic instability of tumor cells (34=E2=80=93= 38) and=20 others that might be shared by many normal cells in the = body. The=20 latter include the lack of expression of costimulatory = molecules=20 (B7-1/CD80, B7-2/CD86, and CD40 ligand), the induction of = suppressor=20 cell activity, and the production of immunoinhibitory = substances=20 such as TGF-=CE=B2 or IL-10. Many of these theories are = intuitively=20 appealing but lack direct experimental evidence or = consistent=20 results. For example, Fas ligand (FasL) has been proposed as = a=20 mediator of the tumor =E2=80=9Ccounterattack.=E2=80=9D = However, controlled=20 experiments show that FasL expressed in animal tumor models = results=20 not in escape, but in more rapid rejection (39=E2=80=93= 42).

Several groups have proposed the loss of=20 =CE=B2₂-microglobulin (=CE=B2₂m) as a = mechanism of=20 immune escape. However, work by Karre, Snook, and colleagues = in=20 animal models demonstrated that the loss of = =CE=B2₂m, an=20 essential and invariant subunit of class I MHC complexes, = results in=20 exquisite sensitivity to NK cell=E2=80=93mediated killing = and leads to tumor=20 elimination, not escape (43,=20 44).=20 Although some human melanoma cells have also been shown to = lose=20 =CE=B2₂m with clinical progression, human=20 =CE=B2₂m=E2=80=93deficient cells are also = susceptible to NK=20 cell=E2=80=93mediated killing (45).=20 This evidence need not indicate that =CE=B2₂m = loss represents=20 a mechanism of immune escape, since this molecule could be = lost as a=20 result of increasing derangement in the transformed genome = and a=20 mutation =E2=80=9Chot spot=E2=80=9D at the = =CE=B2₂m locus (46).=20 Indeed, the mutability of this locus may have unexplored = protective=20 functions. Clearly, similar arguments can be advanced with = regard to=20 other events that decrease or eliminate MHC class I = expression on=20 the surface, such as loss of the MHC class I heavy chain or = of=20 transporters associated with antigen processing or=20 low=E2=80=93molecular-weight proteins complex = components.

To take another example, tumor cells (along with most = normal=20 cells) generally lack costimulatory molecules, such as B7-1 = (CD80)=20 and B7-2 (CD86), which are expressed on professional=20 antigen-presenting cells and on a variety of other tissues = after=20 exposure to inflammatory cytokines (47). In=20 the absence of costimulation, T cells tend to become = anergic. In the=20 non=E2=80=93tumor-bearing setting, the absence of B7 = molecule expression has=20 been hypothesized to protect normal cells against = autoreactivity.=20 Does this finding help explain tumor cell escape from immune = recognition? Transfection of tumor cells with both isoforms = has been=20 used successfully to trigger their immune-mediated rejection = of=20 experimental mouse tumors, which have some inherent = immunogenicity.=20 However, rejection is not observed when B7 molecules are = inserted=20 into less immunogenic tumors (48).=20 Nonimmunogenicity is a category into which most, if not all, = human=20 tumors would fall; thus a lack of expression of the CD80 and = CD86=20 costimulatory molecules is unlikely to be a global = explanation for=20 immune escape. Nonetheless, a greater understanding of the=20 interactions of costimulatory molecules with negative = regulatory=20 molecules, such as cytotoxic T lymphocyte=E2=80=93associated = antigen 4=20 (CTLA-4), may enable more directed interventions (4).

Controlled unresponsiveness and = negative=20 regulation of antitumor T cells

Low-affinity autoreactive T cells can avoid negative = selection in=20 the thymus. Indeed a low level of autoreactivity is required = for=20 positive thymic selection. In normal circumstances, after = maturation=20 is complete, these autoreactive T cells are likely to be = either=20 ignorant (that is, they simply do not =E2=80=9Csee=E2=80=9D = their target epitope) or=20 anergic (defined as a state of induced = unresponsiveness). In=20 the first case, they do not have any contact with the = antigen that=20 alters their phenotype or function. In the latter case, they = are=20 negatively regulated by host factors, such as cytokines, = accessory=20 molecules on antigen-presenting cells, and suppressor cells = (49).=20 Although there is undoubtedly some degree of ignorance to = tumor=20 antigens, there is clear evidence that tumor cells sensitize = host T=20 cells to tumor antigens (our unpublished data).

The spontaneous activation of host tumor-specific T cells = is=20 rarely sufficient to lead to tumor eradication. = Interestingly, a=20 very recent study has revealed a molecular mechanism = mediated by a=20 negative regulatory protein, Tob, a member of an = antiproliferative=20 gene family, in anergized T cells (50).=20 These data may point the way to a new understanding of how = cells=20 maintain unresponsiveness to antigen.

Other candidate negative regulatory mechanisms that may = keep an=20 incipient antitumor response in check include active = negative=20 regulatory mechanisms mediated by = CD4⁺CD25⁺=20 suppressor cells, IL-13=E2=80=93secreting NKT cells, and=20 CD11b⁺Gr-1⁺ suppressor cells. The=20 CD4⁺CD25⁺ T cell population was first = found to=20 inhibit proliferation of = CD4⁺CD25^=E2=80=93 T cells ex=20 vivo as a result of TCR ligation and IL-2 activation. = Extensive=20 studies of suppressor/regulatory T cells in mouse models = have=20 demonstrated their importance in inhibition of autoimmunity = (51).=20 CD4⁺CD25⁺ suppressor T cells also = exist in=20 humans and can inhibit proliferation and cytokine release in = CD4⁺CD25^=E2=80=93 T cells by unknown = mechanisms (52).=20 IL-13 production following CD1 molecule ligation on NKT = cells may=20 also limit antitumor responses. In tumor-bearing mice, CD1 = is=20 upregulated on NKT cells. Knocking out CD1 promotes tumor = resistance=20 in mice. Similar results were also seen in = anti=E2=80=93IL-13=E2=80=93treated mice=20 (53). We=20 do not yet know the relevance of this type of negative = regulation to=20 the immunotherapy of human cancer. Finally, like=20 CD4⁺CD25⁺ T cells and NKT cells,=20 CD11b⁺Gr-1⁺ myeloid cells may send = negative=20 regulatory signals to T cells, triggering apoptotic death in = CD8⁺ T cells following vaccination with powerful=20 immunogens (54).=20 Surprisingly, many mouse and human tumors produce GM-CSF, = which can=20 stimulate highly inhibitory = CD11b⁺Gr-1⁺ cells=20 (55).=20 The manipulation of each of these types of negative = regulatory cells=20 could be useful in the treatment of autoimmune disease and=20 cancer.

Future directions: a focus on T cell = activation=20 and death

Significant evidence indicates that the central reasons = for the=20 failed antitumor immune response may be deficiencies in the=20 maintenance of sustained tumor-specific T cell activation. = It is now=20 clear that there are many ways in which triggering a TCR can = result=20 in the ultimate inactivation or even demise of the T cell = bearing=20 it. The difference between antigen presentation in the tumor = environment and that in a virally infected tissue is likely = the=20 activation of resident antigen-presenting cells, the = scavengers and=20 =E2=80=9Cdanger=E2=80=9D sensors of the immune system. The = lack of proinflammatory=20 mediators that induce maturation of dendritic cells, in = conjunction=20 with the abundant antigen presentation by non-costimulatory, = tolerizing tumor cells, is the factor that may tip the T = cell=20 activation-inactivation balance in favor of tumor-specific T = cell=20 tolerance. On the other hand, overstimulation can terminate = an=20 otherwise effective T cell response through = activation-induced cell=20 death, fratricide, or exhaustion (56).

Although new antigen discovery and epitope mapping = continue to be=20 an important part of tumor immunology, few would dispute = that=20 several excellent targets expressed on a range of tumor = histologies=20 are now available. The next important breakthrough in cancer = immunotherapy may come from an understanding of how to = enhance T=20 cell avidity, how to maintain T cell activation while = preventing T=20 cell apoptosis, and how to reduce or eliminate the effects = of=20 negative regulatory factors.

In animal models, a number of new transgenic mouse models = are now=20 available, allowing for a reductionistic study of tumor = interactions=20 with elements of the innate and adaptive immune system. One=20 particularly fruitful area currently under development = involves the=20 use of TCR transgenic mice. It is now clear that very large = numbers=20 of tumor-specific transgenic CD8⁺ and = CD4⁺ T=20 cells have little effect on the growth rate or lethality of=20 syngeneic tumor cells that express the antigens targeted by = these=20 transgenic T cells. These transgenic mouse systems model key = aspects=20 of increased tumor-specific T cells found in some patients = with=20 cancer after active immunization. Using these models, we and = others=20 are evaluating cellular and molecular mechanisms in T cell=20 activation, death, and anergy as they relate to the = development of=20 more effective cancer vaccines.

Conclusions

Tumor immunologists have made great strides in = understanding the=20 components of the successful immunotherapy of cancer. We = have cloned=20 antigens that are expressed by tumors, processed and = presented in=20 the context of MHC class I and class II molecules, and = recognized by=20 cells from the patient=E2=80=99s own T cell repertoire. We = have also learned=20 how to immunize and are now capable of significantly = expanding=20 precursor T cells with vaccination. Still, the proper and = continued=20 activation of antitumor T cells remains a crucial missing = piece of=20 the immunotherapy puzzle and a significant barrier to = developing an=20 effective therapeutic vaccine. Thus, the focus of tumor=20 immunotherapy is shifting. The challenge now is to learn how = to=20 promote T cell activation and proliferation while abrogating = T cell=20 anergy and death in the context of a profoundly tolerogenic = tumor=20 environment.

References

Rosenberg, SA, et al.Observations on the = systemic=20 administration of autologous lymphokine-activated killer = cells and=20 recombinant interleukin-2 to patients with metastatic = cancer N=20 Engl J Med 1985. 313:1485-1492 3903508[PubMed]=20
Albert, ML, et al.Tumor-specific killer = cells in=20 paraneoplastic cerebellar degeneration Nat Med = 1998.=20 4:1321-1324 9809559[PubMed]=20
Overwijk, WW, et al.Vaccination with a = recombinant=20 vaccinia virus encoding a =E2=80=9Cself=E2=80=9D antigen = induces autoimmune=20 vitiligo and tumor cell destruction in mice: requirement = for=20 CD4(+) T lymphocytes Proc Natl Acad Sci USA 1999.=20 96:2982-2987 10077623[PubMed]=20
van Elsas, A, Hurwitz, AA, Allison, JP, = Combination=20 immunotherapy of B16 melanoma using anticytotoxic T=20 lymphocyte-associated antigen 4 (CTLA-4) and=20 granulocyte/macrophage colony-stimulating factor=20 (GM-CSF)-producing vaccines induces rejection of = subcutaneous and=20 metastatic tumors accompanied by autoimmune depigmentation = J=20 Exp Med 1999. 190:355-366 10430624[PubMed]=20
Cox, AL, et al.Identification of a peptide = recognized=20 by five melanoma-specific human cytotoxic T cell lines=20 Science 1994. 264:716-719 7513441[PubMed]=20
Meunier, L, et al.Quantification of CD1a, = HLA-DR, and=20 HLA class I expression on viable human Langerhans cells = and=20 keratinocytes Cytometry 1996. 26:260-264 = 8979024[PubMed]=20
Parkhurst, MR, et al.Improved induction of=20 melanoma-reactive CTL with peptides from the melanoma = antigen=20 gp100 modified at HLA-A*0201-binding residues J = Immunol=20 1996. 157:2539-2548 8805655[PubMed]=20
Tangri, S, et al.Structural features of = peptide=20 analogs of human histocompatibility leukocyte antigen = class I=20 epitopes that are more potent and immunogenic than = wild-type=20 peptide J Exp Med 2001. 194:833-846 = 11560998[PubMed]=20
Slansky, JE, et al.Enhanced antigen-specific = antitumor=20 immunity with altered peptide ligands that stabilize the=20 MHC-peptide-TCR complex Immunity 2000. = 13:529-538=20 11070171[PubMed]=20
Lyons, DS, et al.A TCR binds to antagonist = ligands=20 with lower affinities and faster dissociation rates than = to=20 agonists Immunity 1996. 5:53-61 8758894[PubMed]=20
Overwijk, WW, et al.gp100/pmel 17 is a = murine tumor=20 rejection antigen: induction of = =E2=80=9Cself=E2=80=9D-reactive, tumoricidal T=20 cells using high-affinity, altered peptide ligand J Exp = Med=20 1998. 188:277-286 9670040[PubMed]=20
Rosenberg, SA, et al.Immunologic and = therapeutic=20 evaluation of a synthetic peptide vaccine for the = treatment of=20 patients with metastatic melanoma Nat Med 1998.=20 4:321-327 9500606[PubMed]=20
Greenberg, PD, Kern, DE, Cheever, MA, = Therapy of=20 disseminated murine leukemia with cyclophosphamide and = immune=20 Lyt-1+,2- T cells. Tumor eradication does not require=20 participation of cytotoxic T cells J Exp Med 1985.=20 161:1122-1134 3921652[PubMed]=20
Surman, DR, Dudley, ME, Overwijk, WW, = Restifo, NP,=20 Cutting edge: CD4+ T cell control of CD8+ T cell = reactivity to a=20 model tumor antigen J Immunol 2000. = 164:562-565=20 10623795[PubMed]=20
Topalian, SL, et al.Human CD4+ T cells = specifically=20 recognize a shared melanoma-associated antigen encoded by = the=20 tyrosinase gene Proc Natl Acad Sci USA 1994.=20 91:9461-9465 7937789[PubMed]=20
Rosenberg, SA, et al.Gene transfer into = humans:=20 immunotherapy of patients with advanced melanoma, using=20 tumor-infiltrating lymphocytes modified by retroviral gene = transduction N Engl J Med 1990. 323:570-578=20 2381442[PubMed]=20
Kim, S, et al.In vivo natural killer cell = activities=20 revealed by natural killer cell-deficient mice Proc = Natl Acad=20 Sci USA 2000. 97:2731-2736 10694580[PubMed]=20
Diefenbach, A, Jensen, ER, Jamieson, AM, = Raulet, DH,=20 Rae1 and H60 ligands of the NKG2D receptor stimulate = tumour=20 immunity Nature 2001. 413:165-171 11557981[PubMed]=20
Ruggeri, L, et al.Effectiveness of donor = natural=20 killer cell alloreactivity in mismatched hematopoietic = transplants=20 Science 2002. 295:2097-2100 11896281[PubMed]=20
Restifo, NP, Vaccines to die for Nat=20 Biotechnol 2001. 19:527-528 11385447[PubMed]=20
Steinman, RM, Pope, M, Exploiting dendritic = cells to=20 improve vaccine efficacy J Clin Invest 2002.=20 109:1519-1526. doi:10.1172/JCI200215962 12070296[PubMed]=20
Ying, H, et al.Cancer therapy using a=20 self-replicating RNA vaccine Nat Med 1999. = 5:823-827=20 10395329[PubMed]=20
Leitner, WW, et al.Enhancement of = tumor-specific=20 immune response with plasmid DNA replicon vectors = Cancer=20 Res 2000. 60:51-55 10646851[PubMed]=20
Palese, P, Zavala, F, Muster, T, = Nussenzweig, RS,=20 Garcia-Sastre, A, Development of novel influenza virus = vaccines=20 and vectors J Infect Dis 1997. 176(Suppl. = 1):S45-S49=20 9240694[PubMed]=20
Restifo, NP, The new vaccines: building = viruses that=20 elicit antitumor immunity Curr Opin Immunol 1996.=20 8:658-663 8902391[PubMed]=20
Rosenberg, SA, et al.Immunizing patients = with=20 metastatic melanoma using recombinant adenoviruses = encoding MART-1=20 or gp100 melanoma antigens J Natl Cancer Inst 1998. = 90:1894-1900 9862627[PubMed]=20
Letvin, NL, Strategies for an HIV vaccine = J Clin=20 Invest 2002. 110:15-20. = doi:10.1172/JCI200215985=20 12093882[PubMed]=20
Kedl, RM, et al.T cells compete for access = to=20 antigen-bearing antigen-presenting cells J Exp Med = 2000.=20 192:1105-1113 11034600[PubMed]=20
Chen, W, Anton, LC, Bennink, JR, Yewdell, = JW,=20 Dissecting the multifactorial causes of immunodominance in = class=20 I-restricted T cell responses to viruses Immunity = 2000.=20 12:83-93 10661408[PubMed]=20
Stoler, DL, et al.The onset and extent of = genomic=20 instability in sporadic colorectal tumor progression = Proc Natl=20 Acad Sci USA 1999. 96:15121-15126 10611348[PubMed]=20
Staveley-O=E2=80=99Carroll, K, et = al.Induction of=20 antigen-specific T cell anergy: an early event in the = course of=20 tumor progression Proc Natl Acad Sci USA 1998.=20 95:1178-1183 9448305[PubMed]=20
Wang, M, et al.Active immunotherapy of = cancer with a=20 nonreplicating recombinant fowlpox virus encoding a model=20 tumor-associated antigen J Immunol 1995.=20 154:4685-4692 7722321[PubMed]=20
McCabe, BJ, et al.Minimal determinant = expressed by a=20 recombinant vaccinia virus elicits therapeutic antitumor = cytolytic=20 T lymphocyte responses Cancer Res 1995. = 55:1741-1747=20 7536130[PubMed]=20
D=E2=80=99Urso, CM, et al.Lack of HLA class = I antigen=20 expression by cultured melanoma cells FO-1 due to a defect = in B2m=20 gene expression J Clin Invest 1991. = 87:284-292=20 1898655[PubMed]=20
Maio, M, Altomonte, M, Tatake, R, Zeff, RA, = Ferrone,=20 S, Reduction in susceptibility to natural killer = cell-mediated=20 lysis of human FO-1 melanoma cells after induction of HLA = class I=20 antigen expression by transfection with B2m gene J Clin = Invest 1991. 88:282-289 1905328[PubMed]=20
Restifo, NP, et al.Molecular mechanisms = used by=20 tumors to escape immune recognition: immunogenetherapy and = the=20 cell biology of major histocompatibility complex class I = J=20 Immunother 1993. 14:182-190 8297900[PubMed]=20
Restifo, NP, et al.Identification of human = cancers=20 deficient in antigen processing J Exp Med 1993.=20 177:265-272 8426105[PubMed]=20
Restifo, NP, et al.Loss of functional beta=20 2-microglobulin in metastatic melanomas from five patients = receiving immunotherapy J Natl Cancer Inst 1996.=20 88:100-108 8537970[PubMed]=20
Restifo, NP, Countering the = =E2=80=98counterattack=E2=80=99=20 hypothesis Nat Med 2001. 7:259 11231598[PubMed]=20
Restifo, NP, Not so Fas: re-evaluating the = mechanisms=20 of immune privilege and tumor escape Nat Med 2000.=20 6:493-495 10802692[PubMed]=20
Restifo, NP, Building better vaccines: how = apoptotic=20 cell death can induce inflammation and activate innate and = adaptive immunity Curr Opin Immunol 2000. = 12:597-603=20 11007365[PubMed]=20
Chappell, DB, Zaks, TZ, Rosenberg, SA, = Restifo, NP,=20 Human melanoma cells do not express Fas (Apo-1/CD95) = ligand=20 Cancer Res 1999. 59:59-62 9892185[PubMed]=20
Kambayashi, T, et al.Purified MHC class I = molecules=20 inhibit activated NK cells in a cell-free system in vitro = Eur J=20 Immunol 2001. 31:869-875 11241292[PubMed]=20
Smyth, MJ, Snook, MB, Perforin-dependent = cytolytic=20 responses in beta2-microglobulin-deficient mice Cell=20 Immunol 1999. 196:51-59 10486155[PubMed]=20
Porgador, A, Mandelboim, O, Restifo, NP, = Strominger,=20 JL, Natural killer cell lines kill autologous=20 beta2-microglobulin-deficient melanoma cells: implications = for=20 cancer immunotherapy Proc Natl Acad Sci USA 1997.=20 94:13140-13145 9371813[PubMed]=20
Parnes, JR, Sizer, KC, Seidman, JG, = Stallings, V,=20 Hyman, R, A mutational hot-spot within an intron of the = mouse beta=20 2-microglobulin gene EMBO J 1986. 5:103-111=20 3514206[PubMed]=20
Anderson, DE, Sharpe, AH, Hafler, DA, The=20 B7-CD28/CTLA-4 costimulatory pathways in autoimmune = disease of the=20 central nervous system Curr Opin Immunol 1999.=20 11:677-683 10631554[PubMed]=20
Chen, L, et al.Tumor immunogenicity = determines the=20 effect of B7 costimulation on T cell-mediated tumor = immunity J=20 Exp Med 1994. 179:523-532 7507508[PubMed]=20
Antony, PA, Restifo, NP, Do CD4+ CD25+=20 immunoregulatory T cells hinder tumor immunotherapy? J=20 Immunother 2002. 25:202-206 12000861[PubMed]=20
Tzachanis, D, et al.Tob is a negative = regulator of=20 activation that is expressed in anergic and quiescent T = cells=20 Nat Immunol 2001. 2:1174-1182 11694881[PubMed]=20
Shevach, EM, Regulatory T cells in = autoimmmunity=20 Annu Rev Immunol 2000. 18:423-449 10837065[PubMed]=20
Dieckmann, D, Plottner, H, Berchtold, S, = Berger, T,=20 Schuler, G, Ex vivo isolation and characterization of=20 CD4(+)CD25(+) T cells with regulatory properties from = human blood=20 J Exp Med 2001. 193:1303-1310 11390437[PubMed]=20
Terabe, M, et al.NKT cell-mediated = repression of=20 tumor immunosurveillance by IL-13 and the IL-4R-STAT6 = pathway=20 Nat Immunol 2000. 1:515-520 11101874[PubMed]=20
Bronte, V, et al.Apoptotic death of CD8+ T=20 lymphocytes after immunization: induction of a suppressive = population of Mac-1+/Gr-1+ cells J Immunol 1998.=20 161:5313-5320 9820504[PubMed]=20
Bronte, V, et al.Unopposed production of=20 granulocyte-macrophage colony-stimulating factor by tumors = inhibits CD8+ T cell responses by dysregulating = antigen-presenting=20 cell maturation J Immunol 1999. = 162:5728-5737=20 10229805[PubMed]=20
Overwijk, WW, Restifo, NP, Creating = therapeutic=20 cancer vaccines: notes from the battlefield Trends = Immunol=20 2001. 22:5-7 11286676[PubMed]=20

Copyright =C2=A9 2008 by the = American Society=20 for Clinical Investigation.
Copying,=20 redistribution, and other usage=20 = policies