Secretomers as a new tool for the monitoring of CTL responses

Efforts to follow tumor-specific immune responses in patients are often thwarted by lack of knowledge of the appropriate tumor antigens and the CTL epitopes of those antigens. There is, therefore, a growing need for techniques to monitor tumor-specific immune responses in settings where tumor antigens, and antigenic epitopes, remain unidentified. Here we describe a novel system to follow tumor-specific CTL immune responses. A truncated, soluble murine class I MHC (H-2D^b) molecule was fused with a rat IgG_2a Fc, in order to allow secretion of the complex. Tumor-specific CTL could then be detected as a result of the complex fastening to specific T cell receptors (TCR). These constructs were inserted into the genome of a recombinant adenovirus vector. Infection of tumor cells with these adenovirus constructs results in the secretion of the complexes into the culture supernatant. These soluble divalent class I MHC molecules were used to detect and activate specific CTL populations.     

Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through "cross-dressing." Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.     

Rejection of mouse sarcoma cells after transfection of MHC class II genes

Th cells are stimulated by peptide Ag presented in the context of MHC class II molecules. We have reasoned that immune responses against tumors may be more efficient if tumor cells were class II Ag positive, and thereby able to directly function as APC to stimulate tumor-specific Th cell proliferation. We have tested this hypothesis by using DNA-mediated gene transfer to generate syngeneic MHC class II Ag-expressing mouse Sal sarcoma cells (Sal/Ak transfectants). Autologous A/J mice challenged i.p. or s.c. with Sal/Ak transfectants do not develop tumors, whereas A/J mice challenged with the class II negative parental Sal tumor have a high tumor incidence. Furthermore, immunization of the autologous host with Sal/Ak transfectants completely protects against subsequent challenge with wild-type Sal cells. MHC class II-expressing tumor cells, therefore, stimulate an improved tumor-specific immune response, and the immunity is cross-reactive with the class II negative tumor. Inasmuch as the transfected MHC class II gene product is not functioning as a target molecule for autologous tumor rejection, the improved immunogenicity of the Sal/Ak cells is probably due to stimulation of a tumor-specific Th cell population. The increased immunogenicity of Sal/Ak cells is, therefore, probably the result of direct presentation of Sal tumor-associated Ag in the context of tumor cell MHC class II molecules to Th lymphocytes. These studies demonstrate that induction of tumor cell MHC class II Ag expression is a potential strategy for tumor-specific immunotherapy, and suggest that tumor immunity may be enhanced by improved Th cell generation.     

Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor's growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.     

An HLA-A2 polyepitope vaccine for melanoma immunotherapy.

Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. Here we describe a recombinant poxvirus vaccine that codes for ten HLA-A2-restricted epitopes derived from five melanoma Ags conjoined in an artificial polyepitope or polytope construct. Target cells infected with the melanoma polytope vaccinia were recognized by three different epitope-specific CTL lines derived from HLA-A2 melanoma patients, and CTL responses to seven of the epitopes were generated in at least one of six HLA-A2-transgenic mice immunized with the construct. CTL lines derived from vaccinated transgenic mice were also able to kill melanoma cells in vitro. Multiple epitopes within the polytope construct were therefore shown to be individually immunogenic, illustrating the feasibility of the polytope approach for melanoma immunotherapy. Tumor escape from CTL surveillance, through down regulation of individual tumor Ags and MHC alleles, might be overcome by polytope vaccines, which simultaneously target multiple cancer Ags.     

Antigenic Differences Between Normal and Malignant Cells as a Basis for Treatment of Intracerebral Neoplasms Using a DNA-Based Vaccine

Antigenic differences between normal and malignant cells of the cancer patient form the rationale for clinical immunotherapeutic strategies. Because the antigenic phenotype of neoplastic cells varies widely among different cells within the same malignant cell-population, immunization with a vaccine that stimulates immunity to the broad array of tumor antigens expressed by the cancer cells is likely to be more efficacious than immunization with a vaccine for a single antigen. A vaccine prepared by transfer of DNA from the tumor into a highly immunogenic cell line can encompass the array of tumor antigens that characterize the patient's neoplasm. Poorly immunogenic tumor antigens, characteristic of malignant cells, can become strongly antigenic if they are expressed by highly immunogenic cells. A DNA-based vaccine was prepared by transfer of genomic DNA from a breast cancer that arose spontaneously in a C3H/He mouse into a highly immunogenic mouse fibroblast cell line, where genes specifying tumor-antigens were expressed. The fibroblasts were modified in advance of DNA-transfer to secrete an immune augmenting cytokine and to express allogeneic MHC class I-determinants. In an animal model of breast cancer metastatic to the brain, introduction of the vaccine directly into the tumor bed stimulated a systemic cellular anti-tumor immune response measured by two independent in vitro assays and prolonged the lives of the tumor-bearing mice. Furthermore, using antibodies against the various T-cell subsets, it was determined that the systemic cellular anti-tumor immunity was mediated by CD8(+), CD4(+) and NK/LAK cells. The application of DNA-based genomic vaccines for the treatment of a variety of brain tumors is being explored.     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

MHC class I antigens,immune surveillance,and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T-lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T-cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T-lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T-cell immunoselection of HLA class I-deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.     

The intrinsic immunogenic properties of cancer cell lines,immunogenic cell death,and how these influence host antitumor immune responses

Modern cancer therapies often involve the combination of tumor-directed cytotoxic strategies and generation of a host antitumor immune response. The latter is unleashed by immunotherapies that activate the immune system generating a more immunostimulatory tumor microenvironment and a stronger tumor antigen-specific immune response. Studying the interaction between antitumor cytotoxic therapies, dying cancer cells, and the innate and adaptive immune system requires appropriate experimental tumor models in mice. In this review, we discuss the immunostimulatory and immunosuppressive properties of cancer cell lines commonly used in immunogenic cell death (ICD) studies being apoptosis or necroptosis. We will especially focus on the antigenic component of immunogenicity. While in several cancer cell lines the epitopes of endogenously expressed tumor antigens are known, these intrinsic epitopes are rarely determined in experimental apoptotic or necroptotic ICD settings. Instead by far the most ICD research studies investigate the antigenic response against exogenously expressed model antigens such as ovalbumin or retroviral epitopes (e.g., AH1). In this review, we will argue that the immune response against endogenous tumor antigens and the immunopeptidome profile of cancer cell lines affect the eventual biological readouts in the typical prophylactic tumor vaccination type of experiments used in ICD research, and we will propose additional methods involving immunopeptidome profiling, major histocompatibility complex molecule expression, and identification of tumor-infiltrating immune cells to document intrinsic immunogenicity following different cell death modalities.Subject terms: Cancer models, Antigen-presenting cells, Immune cell death     

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.     

Analysis of HLA expression in human tumor tissues

Cancer cells can be detected and destroyed by cytotoxic T lymphocytes in many experimental tumor systems, and--as has been well-documented--in some human tumors. In humans however, most diagnosed tumors are not eliminated by T cells but grow steadily, invading and metastasizing until the host is destroyed. Evidence is accumulating that progressive tumor growth occurs not because the immune system is defective or deteriorated, but because the cancer cell is capable of developing a variety of strategies to escape immune recognition. In addition, cancer cells acquire new biological properties to generate invasive capacity in order to migrate and colonize new tissues. Major histocompatibility complex (MHC) antigens are molecules that are specialized in communicating with the T cell receptor and natural killer (NK) cell ligands. With the former, they use the interaction with peptides derived from processed cellular and exogenous proteins to monitor self and non-self status. With the latter, they determine the degree of activation and killing capacity of NK cells by interacting with NK receptors. Any change in the MHC profile of tumor cells (including classical and nonclassical MHC molecules) may therefore have a profound influence on the immune recognition and immune rejection of cancer cells. We have reviewed the data from our laboratory and other groups, and have presented a standardized procedure for analyzing the MHC profile of human tumors with special emphasis on the quality and laboratory use of the material obtained from microdissected tumor samples. Appropriate tissue processing is of particular relevance, since it is not possible to obtain tumor cell lines from most patients. Oncologists require rapid information on the MHC profile of the tumor if gene therapy is envisaged to restore normal MHC class I gene expression.     

Survivin--a universal tumor antigen

Tumor-associated antigens recognized by cellular effectors of the immune system are potential targets for antigen-specific cancer immunotherapy. These antigens are classified as tissue (melanocyte)-specific proteins, cancer-testis antigens (proteins expressed in normal testis and various cancers), tumor-specific peptides derived from mutations in tumor cells, and others. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific cytotoxic T lymphocytes (CTL) responses in vivo. However, most of the peptide epitopes used in these vaccination trials are melanocyte-specific, and these peptides cannot be applied for tumors of non-melanocyte origin. Furthermore, the expression of most tumor antigens is heterogeneous among tumors from different patients and can even vary among metastases obtained from one patient. Immune selection of antigen loss variants may prove to be an additional obstacle for the clinical applicability of most of the known CTL epitopes. Recently, a new tumor antigen, survivin, has been identified on the basis of spontaneous CTL responses in different cancer patients. Survivin is expressed in most human neoplasms, but not in normal, differentiated tissues. Importantly, downregulation or loss of survivin would severely inflict the growth potential of the tumor cell. Since survivin is expressed by a variety of different tumors MHC-restricted survivin epitopes may serve as important and widely applicable targets for anti-cancer immunotherapeutic strategies.     

Oncolytic Immunotherapy: Can’t Start a Fire Without a Spark

Recent advances in cancer immunotherapy have renewed interest in oncolytic viruses (OVs) as a synergistic platform for the development of novel antitumor strategies. Cancer cells adopt multiple mechanisms to evade and suppress antitumor immune responses, essentially establishing a non-immunogenic (‘cold’) tumor microenvironment (TME), with poor T-cell infiltration and low mutational burden. Limitations to the efficacy of immunotherapy still exist, especially for a variety of solid tumors, where new approaches are necessary to overcome physical barriers in the TME and to mitigate adverse effects associated with current immunotherapeutics. OVs offer an attractive alternative by inducing direct oncolysis, immunogenic cell death, and immune stimulation. These multimodal mechanisms make OVs well suited to reprogram non-immunogenic tumors and TME into inflamed, immunogenic (‘hot’) tumors; enhanced release of tumor antigens by dying cancer cells is expected to augment T-cell infiltration, thereby eliciting potent antitumor immunity. Advances in virus engineering and understanding of tumor biology have allowed the optimization of OV-tumor selectivity, oncolytic potency, and immune stimulation. However, OV antitumor activity is likely to achieve its greatest potential as part of combinatorial strategies with other immune or cancer therapeutics.     

Rejection of B16 melanoma induced by expression of a transfected major histocompatibility complex class I gene.

Transfection of a functional major histocompatibility complex class I gene into certain tumor cells, induced by oncogenic viruses or chemical carcinogens, can effectively abrogate their tumorigenic activity. Since experimentally induced tumors possess strong tumor-specific transplantation antigens, expression of cell surface class I antigens may present the tumor cells to appropriate immune effector cells. Most spontaneously arising tumors do not possess tumor-specific transplantation antigens, and their tumorigenicity may not be affected by the expression of a transfected class I gene. We demonstrate that the poorly immunogenic B16-BL6 melanoma can be rendered nontumorigenic in syngeneic mice by the expression of the class I H-2K antigen but not the class II I-A antigen. Furthermore, the poorly tumorigenic, class I-expressing B16-BL6-transfected cells can effectively immunize syngeneic C57BL/6 mice against the highly tumorigenic, class I-deficient B16-BL6 parental cells. Our success in experimentally manipulating the tumorigenicity of a spontaneously derived neoplasm offers hope for a potential modality for the effective treatment of human cancer.     

T-cell death and cancer immune tolerance

Cancer patients mount adaptive immune responses against their tumors. However, tumor develops many mechanisms to evade effective immunosurveillance. T-cell death caused by tumor plays a critical role in establishing tumor immunotolerance. Chronic stimulation of T cells by tumors leads to activation-induced cell death. Abortive stimulation of T cells by tolerogenic antigen-presenting cells loaded with tumor antigens leads to autonomous death of tumor-specific T cells. Therapeutic approaches that prevent T-cell death in the tumor microenvironment and tumor draining lymph nodes, therefore, should boost adaptive immune responses against cancer.     

A ras-mutated peptide targeted by CTL infiltrating a human melanoma lesion

Ags derived from commonly mutated oncogenic proteins seem ideally suited as targets for tumor immunotherapy. Nonetheless, only a few mutated epitopes efficiently presented by human tumors have thus far been identified. We describe here an approach to identify such epitopes. This approach involves: 1) identifying tumors expressing a ras mutation and isolating the tumor-infiltrating lymphocytes (TIL); 2) transfecting COS cells to induce expression of unknown mutated peptides in the context of a patient's HLA class I molecules; and 3) screening epitope recognition by using TIL from the tumors expressing a ras mutation. By using this approach, there appeared to be a N-ras mutation (a glutamine-to-arginine exchange at residue 61 (Q61R)), detected in a melanoma lesion, which was recognized specifically by the autologous TIL in the HLA-A*0101 context. The ras peptide 55-64(Q61R) was the epitope of these TIL and was regularly presented by Q61R-mutated HLA-A*0101(+) melanoma cell lines. This peptide and its wild-type homolog (55-64(wt)) bound to HLA-A*0101 with similar affinities. However, only the mutated peptide could induce specific CTL expansion from PBL. All the CTL clones specific to the mutated peptide, failed to recognize the wild-type sequence on both COS and melanoma cells. These data thus show that oncogenic protein mutations can create shared tumor-specific CTL epitopes, efficiently presented by tumor cells, and that screening for oncogene-transfected COS cell recognition by TIL (from tumors containing mutations) is a powerful approach for the identification of these epitopes.     

CpG-conjugated apoptotic tumor cells elicit potent tumor-specific immunity

The primary goal of cancer immunotherapy is to elicit an immune response capable of eradicating established tumors and preventing tumor metastasis. One strategy to achieve this goal utilizes whole killed tumor cells as the primary immunogen. Killed tumor cells provide a comprehensive source of tumor-associated antigens (TAAs), thereby eliminating the need to identify individual antigens. Unfortunately, killed tumor cells tend to be poorly immunogenic. To overcome this limitation, we covalently conjugated immunostimulatory CpG oligodeoxynucleotides (ODN) to apoptotic tumor cells and examined their ability to induce TAA-specific immune responses. Results indicate that CpG conjugation enhances the uptake of cell-based vaccines by dendritic cells (DCs), up-regulates co-stimulatory molecule expression, and promotes the production of immunostimulatory cytokines. Vaccination with CpG-conjugated tumor cells triggers the expansion of tumor-specific cytotoxic T lymphocytes (CTL) that reduce the growth of established tumors and prevents their metastatic spread. Thus, conjugating CpG ODN to cell-based tumor vaccines is an important step toward improving cancer immunotherapy.