Manipulation of regulatory T cells and antigen-specific cytotoxic T lymphocyte-based tumour immunotherapy.

The most potent killing machinery in our immune system is the cytotoxic T lymphocyte (CTL). Since the possibility for self-destruction by these cells is high, many regulatory activities exist to prevent autoimmune destruction by these cells. A tumour (cancer) grows from the cells of the body and is tolerated by the body's immune system. Yet, it has been possible to generate tumour-associated antigen (TAA) -specific CTL that are also self-antigen specific in vivo, to achieve a degree of therapeutic efficacy. Tumour-associated antigen-specific T-cell tolerance through pathways of self-tolerance generation represents a significant challenge to successful immunotherapy. CD4(+)  CD25(+)  FoxP3(+) T cells, referred to as T regulatory (Treg) cells, are selected in the thymus as controllers of the anti-self repertoire. These cells are referred to as natural T regulatory (nTreg) cells. According to the new consensus (Nature Immunology 2013; 14:307-308) these cells are to be termed as (tTreg). There is another class of CD4(+) Treg cells also involved in regulatory function in the periphery, also phenotypically CD4(+)  CD25(±) , classified as induced Treg (iTreg) cells. These cells are to be termed as peripherally induced Treg (pTreg) cells. In vitro-induced Treg cells with suppressor function should be termed as iTreg. These different Treg cells differ in their requirements for activation and in their mode of action. The current challenges are to determine the degree of specificity of these Treg cells in recognizing the same TAA as the CTL population and to circumvent their regulatory constraints so as to achieve robust CTL responses against cancer.

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.

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.

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.

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.

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.

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.

Inhibition of superoxide generation upon T-cell receptor engagement rescues Mart-1(27-35)-reactive T cells from activation-induced cell death.

Cytotoxic T lymphocytes (CTL) may undergo massive expansion upon appropriate antigenic stimulation. Homeostasis is maintained by a subsequent "contraction" of these cells. Activation-induced cell death (AICD) and programmed cell death prevent the untoward side effects, arising from excessive numbers and prolonged persistence of activated CTL, that occur upon uncontrolled and/or continued expansion. However, effector cell persistence has been identified as a hallmark of successful T-cell-mediated adoptive immunotherapy. Thus, prevention of AICD may be critical to achieve more successful clinical results. We have previously shown that treatment with the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 protects human melanoma epitope Mart-1(27-35)-reactive CTL from apoptotic death upon their reencounter with cognate antigen. However, inhibition of JNK also interferes with the functional ability of the CTL to secrete IFN-gamma. Here, we show that reactive oxygen species (ROS) inhibitors, such as the superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), efficiently protected Mart-1(27-35)-reactive primary CTL from AICD without impairing their functional capability. MnTBAP prevented the increase in intracellular ROS, mitochondrial membrane collapse, and DNA fragmentation observed in control-treated cells upon cognate antigen encounter. Furthermore, the mechanism of AICD prevention in primary CTL included blockade of JNK activation. Finally, tumor-reactive in vitro expanded tumor infiltrating lymphocytes, which are used clinically in cancer immunotherapy, also benefit from MnTBAP-mediated antioxidant treatment. Thus, modulation of the redox pathway might improve CTL persistence and lead to better clinical results for T cell-based immunotherapies.

GITR expression on T-cell receptor-stimulated human CD8 T cell in a JNK-dependent pathway.

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4(+) regulatory T cells and has an important role on cell survival or cell death in CD4(+) T cells. Little is known about the expression of GITR on human CD8(+) T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8(+) T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8(+) T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8(+) T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8(+) T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8(+) cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8(+) T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8(+) cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8(+) cytotoxic T cell response in translational research.

Immunoregulation through IL-10 gene expression and the fate of cytotoxic T lymphocyte-mediated tumor immunotherapy.

Gene analysis of tumor associated antigens revealed that tumor antigens are all normal gene product. Inducing tumor reactive cytotoxic T lymphocytes (CT) in the patients is same as inducing autoimmunity in the patients. Immunosuppressive cytokine interleukin-10 (IL-10) plays an important role in maintaining homeostasis or tolerance. To break the tumor tolerance, blocking and IL-10 secretion or intervention in the pathways of IL-10 gene activation is indeed important.

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.

Presence of low dose of fludarabine in cultures blocks regulatory T cell expansion and maintains tumor-specific cytotoxic T lymphocyte activity generated with peripheral blood lymphocytes.

BACKGROUND: For tumor vaccine-based immunotherapy of cancer, the expansion of tumor antigen-specific cytotoxic T lymphocytes (CTL) in the patients by blocking induced regulatory T (Treg) cells is the most important objective now. Fludarabine (FLU), a known anticancer drug, has been shown to downregulate Treg cells in vivo in chronic leukemia patients. Melanoma tumor antigen Mart-1(27-35)-specific CD8+ CTLs generated in vitrowith total peripheral blood lymphocytes (PBL) lose their activity within 14-21 days with concomitant expansion of Treg cells. When CD4+ cells are removed from PBL and CTL are generated with purified CD8+ cells, the CTL survive and maintain their activity for a significantly longer period. METHODS: We used a low dose of FLU in the cultures in Mart-1-specific CTL generation assays with total PBL. Blood samples were taken from HLA-A2-positive melanoma patients and normal donors. Autologous matured dendritic cells pulsed with Mart-1(27-35) peptide were used to generate CTL responses using purified CD8+ cells or total PBL. RESULTS: The presence of FLU in the cultures with PBL helped to generate a significantly higher number of antigen-specific CTLs as detected by Mart-1 HLA-A2 tetramer staining. Specificity of such CTLs was determined by IFN-gamma secretion or by cytotoxicity against the target cells bearing the specific antigen. The presence of FLU stopped the expansion of IL-10-producing CD4+ Treg cells in the cultures with PBL. Analyses of expanded CD4+ cells isolated from PBL in vitro cocultures with FLU showed a Th1 type of function. Those cells secreted higher amounts of IFN-gamma and very low levels of IL-10, or no IL-10 at all, upon restimulation. CONCLUSION: The observations of the study are as important for adaptive immunotherapy of cancer as they are for vaccine-based approaches.

Silencing of endogenous IL-10 in human dendritic cells leads to the generation of an improved CTL response against human melanoma associated antigenic epitope, MART-1 27-35.

Dendritic cells (DC) present antigenic epitopes to and activate T cells. They also polarize the ensuing T cell response to Th1 or Th2 type response, depending on their cytokine production profile. For example, IL-12 producing DC generate Th1 type T cell response whereas IL-10 producing DC is usually tolerogenic. Different strategies--such as the use of cytokines and anti-cytokine antibodies, dominant negative forms of protein, anti-sense RNA etc.--have been employed to influence the cytokine synthetic profile of DC as well as to make DC more immunogenic. Utilizing GFP expressing recombinant adenoviruses in association with lipid-mediated transfection of siRNA, we have silenced the endogenous IL-10 gene in DC. We show that IL-10 gene silenced DC produces more IL-12 and also generates a better cytolytic T cell response against the human melanoma associated epitope, MART-1(27-35), in vitro. We also show that the GFP expressing adenoviral vector can be used to optimize the parameters for siRNA delivery in primary cells and show that RNA interference methodology can efficiently knock down virus encoded genes transcribed at very high multiplicity of infection in DC.

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 of human melanoma specific cytotoxic T lymphocytes is mediated by apoptosis-inducing factor.

Activation-induced cell death (AICD) of T cells can be an impediment towards achieving a robust and long-lived cytolytic T lymphocyte (CTL) response from active specific immunization or after adoptive cell transfer in cancer immunotherapy. The mechanism of AICD in primary CTL, however, remains poorly understood. It is widely believed that AICD is driven by signals from death receptors (DR) and that the cell death takes place in a caspase-dependent manner, although it has been shown that AICD of T cells can be induced by internal triggers and that death takes place in a caspase-independent manner. We show here that AICD in human melanoma epitope-specific primary CTL involves selective mitochondrio-nuclear translocation of the apoptosis inducing factor (AIF) without cytochrome c release, caspase-3 and caspase-8 activation, and results from large-scale DNA fragmentation. The c-jun-N terminal kinase (JNK) inhibitor, SP600125, blocks the mitochondrio-nuclear translocation of AIF and prevents AICD in these CTL. These findings suggest that the AICD in human melanoma epitope specific primary CTL is mediated by mitochondrial AIF release and JNK is involved in regulation of this death process.

Effect of CD4+CD25+ and CD4+CD25- T regulatory cells on the generation of cytolytic T cell response to a self but human tumor-associated epitope in vitro.

CD4+ T cells naturally expressing CD25 molecules (natural T regulatory cells (Tregs)) have a role in maintaining self tolerance and in regulating responses to infectious agents, transplantation Ags, and tumor Ags. CD4+ Tregs induced from CD4+CD25- precursors (induced Tregs) also regulate immune responses in the periphery. However, which of these Tregs is a major impediment in generating antitumor CTL responses is not clear. We show that although the CD4+CD25+ subsets isolated from peripheral blood-derived lymphocytes do suppress the proliferation of CD4+CD25- effector T cells, they do not suppress the activation and expansion of the self but melanoma-associated, melanoma Ag-reactive T cell 1 (MART-1)27-35-specific CD8+ T cells stimulated by the respective peptide-loaded matured dendritic cells in vitro. The CD4+CD25- counterparts, in contrast, lead to the generation of CD25+ glucocorticoid-inducible TNFR+-Forkhead/winged helix transcription factor+ populations and efficiently suppress the activation and expansion of the MART-127-35 epitope-specific CTLs. Our data suggest that when CTL precursors are optimally stimulated, natural Tregs are not a formidable constraint toward generating a robust antitumor CTL response, but induced Tregs could be.

Macrophages as stimulators of MART-1 27-35 epitope-specific human cytolytic T lymphocytes in vitro.

BACKGROUND: Activation and expansion of antigen-specific cytolytic T lymphocytes (CTL) require epitope presented by antigen-presenting cells (APC). Presently, dendritic cells (DC) are viewed as the most efficient APC. Since the recognition of DCs as the professional APC, the paradigm has emerged that macrophage (MPhi) are scavengers and are incapable of activating T cells. METHOD: The melanoma-associated MART-1(27-35) peptide-loaded MPhi from HLA-A2-positive donors were used to activate MART-1(27-35) epitope-specific CTL in vitro. RESULTS: We show that peptide-pulsed MPhi stimulate MART-1(27-35) epitope-specific precursors to proliferate and to express effector functions. We also show that upon restimulation with the peptide pulsed MPhi, a fraction of the epitope-specific CTLs undergoes activation-induced cell death. The activation-induced cell death is induced in an epitope-specific manner and through apoptosis. CONCLUSION: MPhi can function as APC and are also capable of modulating expansion and contraction of CTL response in vitro.

Continuous presence of Th1 conditions is necessary for longer lasting tumor-specific CTL activity in stimulation cultures with PBL.

The generation of tumor-associated, but self-antigen specific cytotoxic T lymphocytes (CTL) response is possible by vaccination even in patients at the advanced stages of the disease. The in vivo expansion of such CTLs is now the most important objective of the immunotherapy. In human melanoma, we show here that MART-1(27-35)-specific CTLs generated with purified CD8+ cells survive and maintain their activity longer in culture than those CTLs generated by using total peripheral blood lymphocytes (PBL) taken either from patients or from normal donors. When PBL are grown under Th1 conditioning the quantity and quality of CTL with total PBL are comparable with that of the CTLs generated with purified CD8+ cells. For patients either autologous melanoma tumor cells or MART-1(27-35) peptide pulsed autologous DC were used to generate CTL responses. For normal donors MART-1(27-35) peptide pulsed autologous DC were used. For both normal donors or patients, polarization of PBL with Th1 conditioning with interleukin (IL)-12 (250 U/ml) and anti IL-4 antibody 1 mug/ml for 7 days before CTL generation, induced better and longer living CTL response and prevented the expansion of CD4+ T cells that have downregulatory activity. We show that continuous presence of Th1 conditioning in cultures with total PBL generated significantly higher number of antigen-specific CTLs as detected by MART-1 HLA-A2 tetramer staining. The antigen specificity of such CTLs were determined by IFN-gamma secretion in response to target cells bearing the specific antigen. Our observations indicate that Th1 conditioning results in a longer lasting CTL response in vitro and points toward a newer approach for vaccine strategy.

Regulatory T cells and tumor immunity.

Central deletion of "self-reactive" T cells has been the textbook paradigm for inducing "self-tolerance" in the periphery and the concept of a role of T cell-mediated suppression in this process has long been controversial. A decisive shift in the opinion on suppressor T cells has lately occurred with the observations of Sakaguchi's group that linked a class of CD4+CD25+ T cells to the prevention of autoimmunity from neonatal thymectomy in mice. These CD4+CD25+ T cells have been named T regulatory (Treg) cells. They are believed to be selected in the thymus as an anti-self repertoire. Hence they were referred to as natural T regulatory (nTreg) cells. Presently, in addition to their role in autoimmunity, they are believed to exert regulatory function in infection, in transplantation immunity as well as in tumor immunity. In contrast to these nTreg cells, another class of CD4+ Treg cells also exercises regulatory function in the periphery. These Treg cells are also CD4+ T cells and after activation they also become phenotypically CD4+CD25+. They are, however induced in the periphery as Treg cells. Hence, they are termed as induced Treg (iTreg) cells. There are major differences in the biology of these two types of Treg cells. They differ in their requirements for activation and in their mode of action. Nonetheless, evidence indicates that both nTreg cells and iTreg cells are involved in the control of tumor immunity. The question of how to circumvent their regulatory constraints, therefore, has become a major challenge for tumor immunologists.