Chemotherapeutic agents subvert tumor immunity by generating agonists of platelet-activating factor.

Oxidative stress suppresses host immunity by generating oxidized lipid agonists of the platelet-activating factor receptor (PAF-R). Because many classical chemotherapeutic drugs induce reactive oxygen species (ROS), we investigated whether these drugs might subvert host immunity by activating PAF-R. Here, we show that PAF-R agonists are produced in melanoma cells by chemotherapy that is administered in vitro, in vivo, or in human subjects. Structural characterization of the PAF-R agonists induced revealed multiple oxidized glycerophosphocholines that are generated nonenzymatically. In a murine model of melanoma, chemotherapeutic administration could augment tumor growth by a PAF-R-dependent process that could be blocked by treatment with antioxidants or COX-2 inhibitors or by depletion of regulatory T cells. Our findings reveal how PAF-R agonists induced by chemotherapy treatment can promote treatment failure. Furthermore, they offer new insights into how to improve the efficacy of chemotherapy by blocking its heretofore unknown impact on PAF-R activation.

Impaired PIASy-Tip60 signaling weakens activation of p53 in melanoma.

The tumor suppressor p53 plays a central role in preventing tumor development by promoting transcription of genes that stall cell cycle and induce cell death. Although the majority of melanomas express wild-type p53, the molecular mechanisms that impede its activation remain unclear. We previously reported that the SUMO E3 ligase PIASy and the histone acetyltransferase Tip60 signaling cascade promote p53-dependent autophagy and apoptosis. We hypothesized that impairment in this signaling attenuates p53, thus disabling its apoptotic function in melanoma. Here, we show that human melanoma patient samples and cell lines maintain p53 expression but PIASy and/or Tip60 are frequently lost. We observed dysregulation of Tip60-mediated p53 transcription program in melanoma cell lines. Reconstitution of PIASy and Tip60 in melanoma cells increased genotoxic stress-induced apoptosis. Our study provides a clinical link of how sumoylation signaling may activate p53-mediated cell death in melanoma.

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists.

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr -/- mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3(EGFP) transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

Transplantation of mouse HSCs genetically modified to express a CD4-restricted TCR results in long-term immunity that destroys tumors and initiates spontaneous autoimmunity.

The development of effective cancer immunotherapies has been consistently hampered by several factors, including an inability to instigate long-term effective functional antitumor immunity. This is particularly true for immunotherapies that focus on the adoptive transfer of activated or genetically modified mature CD8+ T cells. In this study, we sought to alter and enhance long-term host immunity by genetically modifying, then transplanting, mouse HSCs. We first cloned a previously identified tumor-reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen tyrosinase-related protein 1 (Tyrp1), then constructed both a high-expression lentivirus vector and a TCR-transgenic mouse expressing the genes encoding this TCR. Using these tools, we demonstrated that both mouse and human HSCs established durable, high-efficiency TCR gene transfer following long-term transplantation into lethally irradiated mice transgenic for HLA-DR4. Recipients of genetically modified mouse HSCs developed spontaneous autoimmune vitiligo that was associated with the presence of a Th1-polarized memory effector CD4+ T cell population that expressed the Tyrp1-specific TCR. Most importantly, large numbers of CD4+ T cells expressing the Tyrp1-specific TCR were detected in secondary HLA-DR4-transgenic transplant recipients, and these mice were able to destroy subcutaneously administered melanoma cells without the aid of vaccination, immune modulation, or cytokine administration. These results demonstrate the creation of what we believe to be a novel translational model of durable lentiviral gene transfer that results in long-term effective immunity.

High-avidity autoreactive CD4+ T cells induce host CTL, overcome T(regs) and mediate tumor destruction.

Despite progress made over the past 25 years, existing immunotherapies have limited clinical effectiveness in patients with cancer. Immune tolerance consistently blunts the generated immune response, and the largely solitary focus on CD8+ T cell immunity has proven ineffective in the absence of CD4+ T cell help. To address these twin-tier deficiencies, we developed a translational model of melanoma immunotherapy focused on the exploitation of high-avidity CD4+ T cells that become generated in germline antigen-deficient mice. We had previously identified a tyrosinase-related protein-1 specific HLA-DRB1*0401-restricted epitope. Using this epitope in conjunction with a newly described tyrosinase-related protein-1 germline-knockout, we demonstrate that endogenous tyrosinase-related protein-1 expression alters the functionality of the autoreactive T cell repertoire. More importantly, we show, by using major histocompatibility complex-mismatched combinations, that CD4+ T cells derived from the self-antigen deficient host indirectly triggers the eradication of established B16 lung metastases. We demonstrate that the treatment effect is mediated entirely by endogenous CD8+ T cells and is not affected by the depletion of host regulatory T cells. These findings suggest that high-avidity CD4+ T cells can overcome endogenous conditions and mediate their antitumor effects exclusively through the elicitation of CD8+ T cell immunity.

Tumor-specific Th17-polarized cells eradicate large established melanoma.

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.

Understanding the mechanisms underlying the poor immunogenicity of human self/tumor antigens is challenging because of experimental limitations in humans. Here, we developed a human-mouse chimeric model that allows us to investigate the roles of the frequency and self-reactivity of antigen-specific T cells in determination of the immunogenicity of an epitope (amino acids 209-217) derived from a human melanoma antigen, gp100. In these transgenic mice, CD8+ T cells express the variable regions of a human T cell receptor (hTCR) specific for an HLA-A*0201-restricted gp100(209-217). Immunization of hTCR-transgenic mice with gp100(209-217) peptide elicited minimal T cell responses, even in mice in which the epitope was knocked out. Conversely, a modified epitope, gp100(209-217(2M)), was significantly more immunogenic. Both biological and physical assays revealed a fast rate of dissociation of the native peptide from the HLA-A*0201 molecule and a considerably slower rate of dissociation of the modified peptide. In vivo, the time allowed for dissociation of peptide-MHC complexes on APCs prior to their exposure to T cells significantly affected the induction of immune responses. These findings indicate that the poor immunogenicity of some self/tumor antigens is due to the instability of the peptide-MHC complex rather than to the continual deletion or tolerization of self-reactive T cells.

Immunization of patients with metastatic melanoma using both class I- and class II-restricted peptides from melanoma-associated antigens.

Cancer vaccines targeting CD8+ T cells have been successful in eliciting immunologic responses but disappointing in inducing clinical responses. Strong evidence supports the importance of CD4+ T cells in "helping" cytotoxic CD8+ cells in antitumor immunity. We report here on two consecutive clinical trials evaluating the impact of immunization with both human leukocyte antigen class I- and class II-restricted peptides from the gp100 melanoma antigen. In Protocol 1, 22 patients with metastatic melanoma were immunized with two modified class I A*0201-restricted peptides, gp100:209-217(210M) and MART-1:26-35(27L). In Protocol 2, 19 patients received the same class I-restricted peptides in combination with a class II DRB1*0401-restricted peptide, gp100:44-59. As assessed by in vitro sensitization assays using peripheral blood mononuclear cells (PBMC) against the native gp100:209-217 peptide, 95% of patients in Protocol 1 were successfully immunized after two vaccinations in contrast to 50% of patients in Protocol 2 (P(2) < 0.005). Furthermore, the degree of sensitization was significantly lower in patients in Protocol 2 (P = 0.01). Clinically, one patient in Protocol 2 had an objective response, and none did in Protocol 1. Thus, the addition of the class II-restricted peptide gp100:44-59 did not improve clinical response but might have diminished the immunologic response of circulating PBMC to the class I-restricted peptide gp100:209-217. The reasons for this decreased immune reactivity are unclear but may involve increased CD4+CD25+ regulatory T-cell activity, increased apoptosis of activated CD8+ T cells, or the trafficking of sensitized CD8+ reactive cells out of the peripheral blood. Moreover, the sequential, nonrandomized nature of patient enrollment for the two trials may account for the differences in immunologic response.

Normal tissue depresses while tumor tissue enhances human T cell responses in vivo to a novel self/tumor melanoma antigen, OA1.

Antitumor T cells often recognize targets that are nonmutated "self" tissue differentiation Ags, but the relative impact of Ag expression by normal and transformed tissue for a human self/tumor Ag has not been studied. To examine the influence of self-tolerance mechanisms on the function of self/tumor-specific T cell responses in humans, we sought to identify an Ag that was expressed, processed, and presented in an MHC-restricted fashion by tumor cells, but for which there was the human equivalent of a "knockout." In this study, we report the first immunological characterization of a melanoma/melanocyte differentiation Ag, called OA1, which meets these criteria. This Ag, an X chromosome-encoded melanoma/melanocyte differentiation Ag, was completely deleted in a male patient. Using a newly identified HLA-A*2402-restricted epitope (LYSACFWWL) to study T cell tolerance, we found that OA1-specific T cell reactivity was more than five SD higher in the knockout patient that in normal controls. These data provide compelling evidence for T cell tolerance to OA1 in humans. Most surprisingly, we found elevated levels of OA1-specific T cells in patients with metastatic malignant melanoma, indicating that the tumor-bearing state partially reversed tolerance observed in normal (non-"knockout") individuals. Taken together, these findings indicated that tolerance can exist for self/tumor Ags in humans, and that this tolerance could be partially abrogated by the growth of the tumor, increasing the reactivity of tumor Ag-specific T cells. Thus, the tumor-bearing state reverses, in part, the tolerance of T cells that results from the normal expression of tissue differentiation Ags.

MHC class I and class II presentation of tumor antigen in retrovirally and adenovirally transduced dendritic cells.

The unique antigen-presenting capabilities of dendritic cells (DCs) make them an attractive means with which to initiate an antitumor immune response. Using DCs transduced with tumor antigens for immunotherapy has several theoretical advantages over peptide-pulsed DCs including the possibility that transduced DCs are capable of presenting epitopes on both class I and class II MHC molecules. To test this theory, we inserted the human tumor antigen gp100 into mouse DCs transgenic for HLA-DRbeta1*0401 using either adenoviral vector or a VSV-G pseudotyped retroviral vector. DCs transduced with tumor antigen were able to be recognized by both a murine CD8(+) T-cell clone and a murine CD4(+) T-cell line in a cytokine release assay, thereby demonstrating presentation of both MHC class I and class II gp100 epitopes. This study describes the simultaneous presentation of a tumor-associated antigen to both CD4(+) and CD8(+) T cells and lends support to the use of gene-modified DCs as a means to initiate both CD4(+) and CD8(+) antitumor responses.

Expression of a "self-"antigen by human tumor cells enhances tumor antigen-specific CD4(+) T-cell function.

CD4(+) T cells can recognize "self" tumor antigens, but the impact of tumor cell expression of self-antigens on CD4(+) T-cell function in humans is unknown. Here, we identify a new epitope (ISPNSVFSQWRVVCDSLEDYD) derived from tyrosinase-related protein-1 (TRP-1) using a predictive algorithm and mice transgenic for a chimeric HLA-DRB1*0401 molecule. We then compared the functions of TRP-1-epitope-specific, CD4(+) T-cell responses in normal healthy individuals to those found in patients with metastatic malignant melanoma. Surprisingly, we found that tumor-bearing patients had significantly higher levels of TRP-1-specific, CD4(+) T-cell function than healthy volunteers as measured ex vivo. Thus, the net effect of "self" antigen expression by tumor cells was the enhancement of tumor antigen-specific CD4(+) T-cell function, rather than immunosuppression. These findings indicate that antigens expressed by malignant melanoma cells can partially activate CD4(+) T lymphocytes.

Assumptions of the tumor 'escape' hypothesis.

The reasons why cancer cells are not destroyed by the immune system are likely to be similar, in most cases, to the reasons why normal cells are not destroyed by the immune system. Unfortunately for tumor immunologists, these reasons have not yet been fully elucidated. What is known, however, is that the lack of autoimmune destruction of normal tissue after immune activation is a finely regulated, highly orchestrated sequence of events. Viewed in this light, it is interesting to conceptualize the derangement of the tumor genome not merely as an engine that enables cancer cells to dodge immune recognition. The dysregulation characteristic of the transformed genome is also what makes tumor immunity, a specialized form of autoimmunity, possible.