Current status of chimeric antigen receptor engineered T cell-based and immune checkpoint blockade-based cancer immunotherapies.

Adoptive cell therapies with chimeric antigen receptor (CAR) engineered T cells (CAR-T) and immune checkpoint inhibition (ICI)-based cancer immunotherapies have lately shown remarkable success in certain tumor types. CAR-T cell-based therapies targeting CD19 can now induce durable remissions as well as prolong disease-free survival of patients with CD19 positive treatment refractory B cell malignancies and ICI-based therapies with humanized monoclonal antibodies against the T cell inhibitory receptors CTLA-4 and PD-1 as well as against the PD-1 ligand, PD-L1, can now achieve durable remissions as well as prolongation of life of a sizeable fraction of patients with melanoma and Hodgkin's lymphoma and non-small cell cancers. Most importantly, these immuno-therapeutic treatment modalities have raised the possibility of achieving long-term "containment" as well as "cures" for certain types of cancer. While this represents major advances in cancer immunotherapy, both modalities come with considerable toxicities, including fatalities. Although more work will be needed to bring CAR-T cell-based therapies to the bedside for most major cancers and a good deal more will be needed to make ICI-alone or in combination with other treatment modalities-work more consistently and across most major cancers, these two treatment modalities stand out as superb examples of successful translation of bench research to the bedside as well as represent real progress in the field of cancer immunotherapy.

Knockdown of T-bet expression in Mart-127-35 -specific T-cell-receptor-engineered human CD4(+) CD25(-) and CD8(+) T cells attenuates effector function.

Gene transfer to create tumour epitope-specific cytolytic T cells for adoptive immunotherapy of cancer remains an area of active inquiry. When the Mart-127-35 -specific DMF5 T-cell receptor (TCR) is transferred into peripheral human CD4(+) T cells, the reprogrammed cells exhibit a T helper type 1 (Th1) phenotype with significant multifactorial effector capabilities. The T-bet transcription factor plays an important role in determination of the Th1 differentiation pathway. To gain a deeper understanding of how T-bet controls the outcome of human T-cell reprogramming by gene transfer, we developed a system for examining the effects of short hairpin RNA-mediated T-bet gene knockdown in sorted cell populations uniformly expressing the knockdown construct. In this system, using activated peripheral human CD4(+)  CD25(-) and CD8(+) T cells, T-bet knockdown led to attenuation of the interferon-γ response to both antigen-specific and non-specific TCR stimulation. The interleukin-2 (IL-2) antigen-specific response was not attenuated by T-bet knockdown. Also, in TCR-reprogrammed CD8(+) cells, the cytolytic effector response was attenuated by T-bet knockdown. T-bet knockdown did not cause redirection into a Th2 differentiation pathway, and no increased IL-4, IL-10, or IL-17 response was detected in this system. These results indicate that T-bet expression is required for maintenance of the CD4(+)  CD25(-) and CD8(+) effector phenotypes in TCR-reprogrammed human T cells. They also suggest that the activation protocol necessary for transduction with retrovectors and lentivectors may commit the reprogrammed cells to the Th1 phenotype, which cannot be altered by T-bet knockdown but that there is, nevertheless, a continuous requirement of T-bet expression for interferon-γ gene activation.

Death receptor-independent activation-induced cell death in human melanoma antigen-specific MHC class I-restricted TCR-engineered CD4 T cells.

Engaging CD4 T cells in antitumor immunity has been quite challenging, especially in an Ag-specific manner, because most human solid tumors usually do not express MHC class II molecules. We have recently shown that human CD4 T cells engineered to express a human melanoma-associated antigenic epitope, MART-127-35, specific MHC class I-restricted transgenic TCR function as polyfunctional effectors that can exhibit a helper as well as cytolytic effector function, in an epitope-specific and MHC class I-restricted manner (Chhabra et al. 2008. J. Immunol. 181: 1063-1070; Ray et al. 2010. Clin. Immunol. 136: 338-347). TCR-engineered (TCReng) CD4 T cells therefore have translational potential, and clinical trials with MHC class I TCReng CD4 T cells are under way. In this study, we show that although TCReng CD4 T cells could be useful in cancer immunotherapy, they are also susceptible to epitope-specific activation-induced cell death (AICD). We also show that the AICD in TCReng CD4 T cells is a death receptor-independent process and that JNK and p53 play critical roles in this process as pharmacological inhibitors targeting JNK activation and p-53-mediated transcription-independent mitochondria-centric death cascade rescued a significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function. Our data offer novel insights toward AICD in TCReng CD4 T cells and identify several potential targets to interfere with this process.

T cells expanded in presence of IL-15 exhibit increased antioxidant capacity and innate effector molecules.

Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. Whereas long-term IL-2 exposure has been shown to promote apoptosis and limit CD8(+) memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and helps maintain a memory CD8(+) T-cell population. However, mechanisms for superior outcomes for IL-15 as compared to IL-2 are still under investigation. Our data shows that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules glutathione reductase (GSR), thioredoxin reductase 1 (TXNDR1), peroxiredoxin (PRDX) and superoxide dismutase (SOD). An increased expression of cell-surface thiols, intracellular glutathione, and thioredoxins was also noted in IL-15 cultured T cells. Additionally, IL-15 cultured T cells showed an increase in cytolytic effector molecules. Apart from increased level of Granzyme A and Granzyme B, IL-15 cultured T cells exhibited increased accumulation of reactive oxygen (ROS) and reactive nitrogen species (RNS) as compared to IL-2 cultured T cells. Overall, this study suggests that T cells cultured in IL-15 show increased persistence not only due to levels of anti-apoptotic proteins, but also due to increased anti-oxidant levels, which is complimented by increased cytolytic effector functions.

The oxazolidinone derivative locostatin induces cytokine appeasement.

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

MHC-I-restricted melanoma antigen specific TCR-engineered human CD4+ T cells exhibit multifunctional effector and helper responses, in vitro.

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.

CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model.

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.

Inhibition of c-Jun N-terminal kinase rescues influenza epitope-specific human cytolytic T lymphocytes from activation-induced cell death.

Cytolytic T lymphocytes (CTL) play an important role in defense against viral infections. Following clonal expansion and effector functions, a vast majority of the antigen-specific CTL undergoes programmed cell death to maintain homeostasis. We have shown earlier that melanoma epitope-specific CTL are quite sensitive to activation-induced cell death (AICD) even on the secondary encounter of the antigen. Excessive sensitivity of viral antigen-specific CTL to AICD, however, would be counterproductive. It might be argued that although CTL for a "self" epitope might be more prone to AICD for maintaining self-tolerance, viral antigen-specific CTL are likely to be less sensitive to AICD. We show here that influenza matrix protein-derived MP(58-66) epitope-specific CTL, activated in vitro and bearing a memory phenotype, are just as sensitive to AICD. The AICD in these CTL is not blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone or by soluble Ig-Fc chimeras of the death receptors [Fas, TNF receptor (TNF-R), TRAIL-RI, TRAIL-RII]. However, the MP(58-66)-specific CTL can be rescued from AICD by the c-jun-N-terminal kinase (JNK) inhibitor SP600125. These results have implications for immunotherapeutic intervention in rescuing viral epitope-specific CTL from AICD.

Activation induced cell death (AICD) of human melanoma antigen-specific TCR engineered CD8 T cells involves JNK, Bim and p53.

OBJECTIVES: Adoptive cancer immunotherapy (ACT) with transgenic T cell receptor (TCR) engineered (TCReng) anti-tumor T cells has produced encouraging results, however, efficacy of these approaches need improvement. Since premature activation induced cell death (AICD) of adoptively administered T cells could be a major impediment, we examined the mechanism(s) underlying AICD in TCReng CD8+ cytolytic T lymphocytes (CTL). METHODS: AICD in human tumor antigen-specific MHC class I restricted TCR engineered CD8+ CTL was induced by exposing them to cognate peptide epitope. RESULTS: We show that TCReng CD8+ human primary CTL undergo AICD even upon encountering their cognate peptide epitope for the very first time. AICD in TCReng CTL is a death-receptor-independent, JNK activation-driven intrinsic processes, in which p53-mediated mitochondria-centric, non-transcription-dependent pathway plays an essential role. Activated JNK modulates mitochondrial membrane integrity in CTL undergoing AICD by directly interacting with Bcl family protein, Bim, and the mitochondrial membrane pore complex, voltage dependent anion channel (VDAC), leading to the release of caspase-independent death executioner, apoptosis inducing factor (AIF), accumulation of single strand DNA breaks and eventually to cell death. CONCLUSIONS: Our findings offer opportunities to interfere with AICD in TCReng CD8+ anti-tumor CTL for sustaining them longer for producing better clinical outcomes.

Regulation of melanoma epitope-specific cytolytic T lymphocyte response by immature and activated dendritic cells, in vitro.

Dendritic cell (DC)-based immunization in cancer has proven to be a promising approach. However, just as DCs are crucial accessory cells in generating immune responses, they also seem to participate in tolerance induction, especially against peripheral "self" antigens. The bulk of the evidence that DCs present peripheral self antigens to induce tolerance has, however, come mostly from studies in transgenic animal models. A tolerogenic function of DCs for peripheral self antigens in a human model has not been critically examined. In this study using the Melan-A/MART-1(27-35) peptide as a model for self but melanoma-associated antigen-against which human hosts often harbor CD8(+) CTL precursors with high frequencies-we confirm that although immature dendritic cells (iDCs) are inefficient antigen presenting cells (APCs), fully activated DCs efficiently activate melanoma epitope-specific CD8(+) CTL precursors, in vitro. We, however, show that in a direct epitope presentation schema, iDCs neither delete nor anergize epitope-specific CD8(+) T cells in primary or secondary stimulation. Interestingly, iDCs and activated DCs can delete a large fraction of the epitope-specific CTLs on tertiary stimulation. The deletion is induced in an epitope-specific manner and through apoptosis. These observations, therefore, have implications on the DC-based cancer vaccine designs and are relevant in the inquiry into the role of DCs on tolerance induction.

Suppression of inducible CD4 regulatory cells by MHC class I-restricted human tumor epitope specific TCR engineered multifunctional CD4 T cells.

Regulatory T cells (Treg) can interfere with the generation and function of anti-tumor immune effectors. Accordingly, ways that could block Treg function would be useful in cancer immunotherapy. We have previously shown that incorporation of CD4+CD25-ve T cells in an in vitro cytolytic T lymphocyte (CTL) generation assay leads to generation of induced regulatory T cells (iTregs), and that these iTreg block the generation of productive CTL response (Chattopadhyay et al., 2006). We here show that human CD4 T cells engineered to express MHC class I-restricted human melanoma associated epitope, MART-127-35, specific T cell receptor (TCR), that can simultaneously exhibit helper as well as cytolytic effector functions (Chhabra et al., 2008, Ray et al., 2010), can interfere with the generation of inducible Treg, block iTreg-mediated suppression, and allow the activation and expansion of MART-127-35 specific CTL responses, in vitro. We also show that mitigation of Treg generation by TCR engineered CD4 T cells is not mediated by a soluble factor and may involve "licensing/conditioning" of the dendritic cells (DC). Our data offer novel insights on the biology of MHC class I restricted TCReng CD4 T cells and have translational implications.

Medical Education: The Hot Seat.

Medical science has eventually metamorphosed from 'Knowledge based' to 'Skill based' applied social science. So, the age-old traditional courses and curriculums in Indian medical education need a overhauling with radical modifications. With a paradigm shift, we have to take into account not only the help of scientific feedback from the teachers and students but also from all the stakeholders of health care delivery system.

Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma.

PURPOSE: The purpose of this study was to determine the toxicity and immunological effects of three different doses and two routes of administration of autologous dendritic cells (DCs) pulsed with the MART-1(27-35) immunodominant epitope. EXPERIMENTAL DESIGN: Eighteen HLA-A*0201-positive subjects with stage III-IV melanoma received three biweekly i.v. or intradermal injections of ex vivo generated myeloid DCs pulsed with MART-1(27-35) epitope. Repeated blood samples were processed to obtain peripheral blood mononuclear cells for immunological analysis using IFN-gamma ELISPOT, MHC class I tetramer, intracellular cytokine staining, and microcytotoxicity assays. RESULTS: The frequency of MART-1/Melan-A (MART-1) antigen-specific T cells in peripheral blood increased in all dose levels as assessed by ELISPOT and MHC class I tetramer assays, but without a clear dose-response effect. The intradermal route generated stronger MART-1 immunity compared with the i.v. route. MART-1-specific immunity did not correlate with clinical outcome in any of the four immunological assays used. However, analysis of determinant spreading to other melanoma antigens was noted in the only subject with complete response to this single-epitope immunization. CONCLUSIONS: Intradermal immunization with MART-1 peptide-pulsed DCs results in an increase in circulating IFN-gamma-producing, antigen-specific T cells. The frequency of these cells did not correlate with response. In contrast, spreading of immune reactivity to other melanoma antigens was only evident in a subject with a complete response, suggesting that determinant spreading may be an important factor of clinical response to this form of immunotherapy.

Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma.

A role of CD4(+) cells in the regulation of immune responses has steadily gained renewed recognition. The understanding of these T-regulatory (T-reg) cells in the generation of antitumor cytolytic T lymphocyte (CTL) response is therefore important. It has been shown that immunization with specific peptides, DNA, or tumor lysate-based vaccines can induce CTL responses in vivo. We have immunized melanoma patients with major histocompatibility complex (MHC) class I restricted peptide- or melanoma tumor lysate-loaded antigen-presenting cell (APC)-based vaccines and have monitored the generation of CTL responses and T-reg cell responses, if any. Using tetramer staining and limiting dilution analyses as monitors of CTL responses, we found significant increases in the number of antigen-specific CTL in circulation after vaccination with the MART-1(27-35) peptide (AAGIGILTV)-pulsed autologous APC, the MAGE-1(161-169) peptide (EADPTGHSY)-pulsed APC, or with autologous tumor lysate-pulsed APC. The antigen-specific CTL reached the peak expansion by day 7 and then declined to the prevaccine levels by day 28. The decline in the CTL response was associated by a concomitant expansion of CD4(+) CD25(+)T cells. Analysis of postvaccine peripheral blood lymphocytes (PBL) from patients showed an increased amount of interleukin (IL)-10 secretion on in vitro stimulation with IL-2 after successive vaccination. Triple color flow cytometric analyses revealed cytoplasmic IL-10 in the CD4(+)CD25(+) T-cell fraction and the number of CD4(+)CD25(+) IL-10(+) T cells were found to increase significantly in postvaccine PBL. These observations have implications in tumor antigen and APC/dendritic cell (DC)-based cancer vaccine strategies.

Adoptive transfer of MART-1 T-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma.

PURPOSE: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy. EXPERIMENTAL DESIGN: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation. RESULTS: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells. CONCLUSION: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.

Immunology of melanoma.

It can be safely said that human melanomas are immunogenic. Virtually all the major principles of "tumor immunology" have been experimentally established in this model. It is now amply clear that melanoma cells display multiple antigens and peptide epitopes that are targetable by the host immune system and that patients with melanoma are capable of responding to these antigens and epitopes serologically as well as through the cell-mediated mechanisms. The immune responses against melanoma are, however, subject to regulation by the regulatory processes within the immune system itself and melanoma cells can resort to overt evasive activities. Indeed, the intrinsic as well as the extrinsic mechanisms within the immune system that are designed to control the magnitude as well as the duration of immune responses at times act as constraints against generating a robust and long-lasting antimelanoma response and melanoma cells are capable of using all the tricks (eg, downregulation of targetable molecules, elaboration of immunosuppressive cytokines) available to living organisms so as to evade immune recognition and destruction. As a result, the immune system often fails to protect the host against melanoma development and progression. The cumulative knowledge over the years on melanoma-associated antigens and epitopes, on methods of immunization, and on technologies for generating melanoma antigen-specific T cells, natural or engineered, have led to the development of immunotherapeutic strategies with "melanoma vaccines" and with T-cell-based adoptive immunotherapy for melanoma. Although these strategies have not been uniformly successful in all cases, durable complete regressions of metastatic melanoma can at times be obtained with active specific immunization or adoptive cell therapy. There is reason for hope that continued research in the field is likely to improve the outcome of melanoma immunotherapy: the ultimate goal of tumor immunology.

Analyses of T cell-mediated immune response to a human melanoma-associated antigen by the young and the elderly.

Elderly cancer patients are often excluded from immune-based clinical trials and therapies based on the belief that they respond poorly to tumor antigens. Using melanoma as a model and melanoma related Mart-127-35 epitope specific T cell receptor (TCR) engineered T cells as a tool we compared the T cell responses from young and elderly to the Mart-127-35 epitope, ex vivo. We also compared the natural Treg (nTreg) activities and the expression of a number of genes associated with immune response by quantitative real-time reverse Transcription Polymerase Chain Reaction (qRTPCR) in formalin fixed primary melanomas, in situ. We detected a significant difference in CD8(+) T cell response to Flu antigen (influenza matrix peptide Flu MP58-66), but the responses of the two cohorts to melanoma antigen were comparable. nTreg activities in the elderly was significantly compromised. The qPCR analyses of tissues from elderly patients revealed lower levels of Fox-P3 expression but comparable levels of expression of IL-2, IFNγ, TNFα, IL-4, IL-10, IDO, and TGFß. These findings indicate that elderly patients might be capable of responding to tumor antigens, and need not be excluded from immune-based therapies or clinical trials.

Metastatic melanoma in the older patient: immunologic insights and treatment outcomes.

Cutaneous melanoma (CM) is a highly curable skin cancer of melanocytes if diagnosed early. Unfortunately, its invasion into the deeper dermis increases the risk of it spreading to the lymph nodes and distant organs. Spread of metastatic melanoma (MM) to other organs is among one of the most dangerous conditions that is almost uniformly fatal for the majority of patients with the currently available treatment modalities. Since melanoma is an immunogenic tumor, developing novel immune strategies will continue to play a critical role in designing effective treatment modalities for those at high risk of recurrence and those with distant metastasis. While older age is believed to be a poor prognostic marker for CM, rapid expansion of the aging population and its projected increase in the coming decades is expected to result in a large number of elderly melanoma patients seeking treatment in all stages of disease. This will not only bring with it unique management challenges in this population, but also an increased burden on communities to provide financial and social resources. Comprehensive efforts will need to be directed towards early diagnosis, as well as developing safe and effective treatment. Renewed interest in the cancer immune surveillance theory coupled with recognition of aging-associated weaknesses in the immune system has put the spotlight on immunsenescence as a important risk factor for the rising incidence of CM in the aging population. Comprehensive assessment of the aging immune system might shed light, not only on weaknesses of individual components of the adaptive immune system, but also on the critical imbalances resulting from these weaknesses on anti-melanoma immunity. Identifying these imbalances might help harness novel immune-based treatment of MM in selected elderly patients. This article describes our experience of treating elderly patients with MM and the issues unique to them, with particular emphasis on insights into the aging immune system.

Obstacles to and opportunities for more effective peptide-based therapeutic immunization in human melanoma.

Melanoma cells can play a number of tricks to evade the host immune response. They can make themselves invisible to cells of the immune system poised to attack them, elaborate molecules that are frankly immunosuppressive, and can create a microenvironment that is hostile to cells of the immune system. Efforts are underway to institute measures that would make tumor cells more susceptible to immune attack, but these efforts have not been all that successful so far. This contribution reviews the history and the rationale of cancer vaccines, the major obstacles to peptide-based immunization, and a discussion on how to surmount them. Also included are the roles played by peripheral tolerance, low-affinity T-cell receptors, T-cell ignorance, activation-induced cell death, exhaustion of T cells and regulation of the immune response, helpless cytolytic T lymphocytes, and evasion by tumor cells.