Human memory T cells present in ovarian tumor ascites fluids fail to respond normally to stimulation via the T cell receptor (TCR). This immunosuppression is manifested by decreases in NF-κB and NFAT activation, IFN-γ production, and cell proliferation in response to TCR stimulation with immobilized antibodies to CD3 and CD28. The anergy of the tumor-associated T cells (TATs) is mediated by soluble factors present in ovarian tumor ascites fluids. The non-responsiveness of the T cells is quickly reversed when the cells are assayed in the absence of the ascites fluid, and is rapidly reestablished when a cell-free ascites fluid is added back to the T cells. Based upon the observed normal phosphorylation patterns of the TCR proximal signaling molecules, the inhibition of NF-κB, and NFAT activation in response to TCR stimulation, as well as the ability of the diacylglycerol analog PMA and the ionophore ionomycin to bypass the ascites fluid-induced TCR signaling arrest, the site of the arrest in the activation cascade appears to be at or just upstream of PLC-γ. An identical TCR signaling arrest pattern was observed when T cells derived from normal donor peripheral blood were incubated with either malignant or nonmalignant (cirrhotic) ascites fluids. The immunosuppressive activity of ascites fluids reported here suggests that soluble factors acting directly or indirectly upon T cells present within tumors contribute to the anergy that has previously been observed in T cells derived from malignant and nonmalignant inflammatory microenvironments. The soluble immunosuppressive factors represent potential therapeutic targets for ovarian cancer.
The plethora of tumor antigens that have been--and are still being--defined required systematization to provide a comprehensive overview of those tumor antigens that are the most relevant targets for cancer immunotherapy approaches. Here, we provide a new update of a peptide database resource that we initiated many years ago. This database compiles all human antigenic peptides described in the literature that fulfill a set of strict criteria needed to ascertain their actual "tumor antigen" nature, as we aim at guiding scientists and clinicians searching for appropriate cancer vaccine candidates (www.cancerimmunity.org/peptide). In this review, we revisit those criteria in light of recent findings related to antigen processing. We also introduce the 29 new tumor antigens that were selected for this 2013 update. Two of the new peptides show unusual features, which will be briefly discussed. The database now comprises a total of 403 tumor antigenic peptides.
Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to quantify changes in the tumor vasculature, and to demonstrate and study the immunosuppressive effects of the tumor microenvironment. Using the OTX model, we show that the tumor-associated T cells originally present within the tumor tissues are anergic and that fully functional autologous T cells injected into tumor-bearing mice localize within the tumor xenograft. The transferred T cells remain functional for up to 3 days within the tumor microenvironment but become unresponsive to activation after 7 days. The OTX model provides for the first time the opportunity to study in vivo the cellular and molecular events contributing to the arrest in T cell function in human ovarian tumors.
Cancer/Testis (CT) antigens are normally only expressed in germ cells and yet are aberrantly activated in a wide variety of human cancers. Most chromosome X-encoded CT antigens (CT-X) show restricted expression in pre-meiotic germ cells in adult testis, except for the expression of SPANX in post-meiotic germ cells. In the present study, the expression of eight CT-X antigens (MAGE-A, NY-ESO-1, GAGE, MAGE-C1/CT7, MAGE-C2/CT10, CT45, SAGE1, and SPANX) in non-seminomatous germ cell tumors was evaluated immunohistochemically, including 24 embryonal carcinomas, 20 yolk sac tumors, 9 teratomas, and 3 choriocarcinomas, and the results were compared to our previous study of 77 classic seminomas and 2 spermatocytic seminomas. SPANX was not detected in any germ cell tumors tested. Spermatocytic seminoma showed strong expression of all CT-X antigens tested (except SPANX), reflecting their origin from adult CT-Xpositive pre-meiotic germ cells. Classic seminomas, originating from prenatal gonocytes, showed widely variable frequency of CT-X antigen expression, ranging from > 80% (CT7, CT10, CT45, and GAGE), 63% (MAGE-A), 18% (NY-ESO-1) to only 4% (SAGE1). In comparison, non-seminomatous germ cell tumors expressed CT-X antigens much less frequently and usually only in small subsets of tumor cells. Intratubular germ cell neoplasia (ITGCN) were mostly CT-X-negative, even in CT-X positive classic seminomas. These findings indicate that CT-X antigens are not expressed in the fetal precursor cells for germ cell tumors, and their expression likely reflects germ cell differentiation of the neoplastic cells (in seminomas) or aberrant gene activation as cancer antigens (in non-seminomatous tumors).
Invariant natural killer T (iNKT) cells are innate T lymphocytes that specifically recognize α-linked glycosphingolipids (α-GSLs) as antigens presented by CD1d molecules. Activating iNKT cells by administering α-GSLs improves disease outcomes in murine cancer models and, thus, there is great interest in the clinical potential of these lipids for treating human cancers. However, humans possess several other CD1 isoforms that are not present in mice and it is not clear whether these CD1 molecules, which also bind lipids, affect human iNKT cell responses. We demonstrate here that CD1c, which is co-expressed with CD1d on blood dendritic cells and on a fraction of B cells, is able to present α-galactosylceramide (α-GalCer) as a weak agonist to human iNKT cells, and that the presence of CD1c synergistically enhances α-GalCerdependent activation of iNKT cells by CD1d. Primary human B cells expressing CD1c induced stronger iNKT cell responses to α-GalCer than the CD1c- subset, and an antibody against CD1c inhibited iNKT cell cytokine secretion. These results suggest that therapeutic activation of human iNKT cells by α-GSLs will be driven preferentially by CD1c+ cell types. Thus, B cell neoplasias that co-express CD1c and CD1d may be particularly susceptible to α-GSL therapy, and cancer vaccines using α-GSLs as adjuvants may be most effective when presented by CD1c+ antigen-presenting cells.
Recently, the final versions of the Minimal Information about T cell assays (MIATA) guidelines were published following a vetting process among peers. Here we summarize the rationale and background of the project and propose how to implement structured reporting of T cell experiments. Successful adoption by peers bears the potential, in the long-term, of enhancing the development of new immunomodulatory therapeutics. Assays for the analysis of cell-mediated immunity belong to the essential repertoire of many laboratories involved in a variety of immunological sub-specialties, including oncology, infectious diseases, autoimmunity, transplantation immunology, and others. Flow cytometry-based as well as Elispot assays have been developed to investigate a wide range of analytes on a single-cell level and were refined for sensitive, reliable, and polyfunctional analysis. Over the years, the assay evolution occurring in institutions worldwide has led to optimized assays, but has left the scientific community with countless permutations of assay protocols and local standards. At the institutional level, the two main challenges are to define the biomarker assay which fits best to the investigational medicinal product in development, and to control the performance of applied analytical assays. From the community-wide perspective, the biggest challenge arises from the question of how to reliably interpret and compare results generated by different labs. In this dynamic setting, the Cancer Immunotherapy Consortium of the Cancer Research Institute (CIC-CRI) and the Immunoguiding Program of the Association for Cancer Immunotherapy (CIP-CIMT) initiated proficiency panel programs for the most commonly used T cell assays involving a large number of heterogeneous labs from different backgrounds (1). These panels revealed that results from testing the same samples can vary significantly between labs (2, 3). Hence, the heterogeneous landscape of T cell immune assays not only is reflected by the use of different protocols, but also directly translates into wide-spread variability in assay results. Data obtained from these large-scale proficiency panels enabled the identification of critical variables in assay design and conduct that can influence assay results (Figure 1). Figure 1 Examples for sources of variation for cellular assays. All of these variables have been shown to impact assay performance. Crucial findings from these efforts were summarized in harmonization guidelines for the community (3–6). In subsequent panels, the implementation and adherence to such harmonization guidelines were successful in improving the accuracy of assay results overall and reducing the variability among labs (7). However, it was noted early on that most publications on T cell-related immune monitoring data lack structured reporting of all the critical protocol variables that may influence assay results, which may prevent easy interpretation of reports and limit comparability of data generated across institutions. To address this issue, the Minimal Information About T Cell Assays (MIATA) project was initiated to define reporting guidelines, mirroring similar Minimal Information (MI) projects that were initially developed for high-throughput genomic assays and successfully applied for a variety of other assays (2, 8, 9). Importantly, MIATA was started and carried out as a broad effort to reach consensus on the minimal information necessary to efficiently and transparently describe how T cell assays were performed such that peers can confidently understand and interpret the presented data. While this was the project’s driving force, another question of similar significance to be asked was: what and how much are scientists willing to share? Consequently, the MIATA project included an intense vetting process with two public consultation periods and two open workshops, with constant outreach to the community, over the time frame of three years (10). A dedicated website (11) was created that comprehensively displays every step, comment, and participant contributing to the project. With the input of more than 120 peers from academia and industry, as well as from regulatory background, the MIATA guidelines were recently finalized (12). The guidelines are divided into five modules and additional sub-modules which relate to the information concerning the sample, the specific assay protocol, the data acquisition, and analysis, as well as the lab environment—all process parameters identified earlier to be critical variables that can influence assay results. The guidelines are also visible on the MIATA website.
During cancer progression, malignant cells may evade immunosurveillance. However, evidence for immunological escape in humans is scarce. We report here the clinical course of a melanoma patient whose initial tumor was positive for the antigens NY-ESO-1, MAGE-C1, and Melan-A. Upon immunization with a recombinant vaccinia/fowlpox NY-ESO-1 construct, the patient experienced a mixed clinical response and spreading of the NY-ESO-1 epitopes in the CD4+ T cell compartment. After NY-ESO-1 protein + CpG immunization, the patient's anti-NY-ESO-1 IgG response increased. Over the following years, progressing lesions were resected and found to be NY-ESO-1-negative while being positive for MAGE-C1, Melan-A, and MHC-I. The fatal, inoperable brain metastasis was analyzed after his death and also proved to be NY-ESO-1-negative, while being positive for MAGE-C1 and Melan-A, as well as MHC-I. We propose that cancer control and cancer escape in this patient were governed by NY-ESO-1-specific immunological pressure. Our findings provide evidence for the existence of immunoediting and immunoescape in this cancer patient.
We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in "humanized" mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells.
The Natural Killer Group 2D (NKG2D) receptor plays an important role in protecting the host from infections and cancer. By recognizing ligands induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells. Because these ligands are not widely expressed on healthy adult tissue, NKG2D ligands may present a useful target for immunotherapeutic approaches in cancer. Novel therapies targeting NKG2D ligands for the treatment of cancer have shown preclinical success and are poised to enter into clinical trials. In this review, the NKG2D receptor and its ligands are discussed in the context of cancer, infection, and autoimmunity. In addition, therapies targeting NKG2D ligands in cancer are also reviewed.
We describe a randomized three-arm phase I study of ipilimumab administered alone (I group) or in combination with dacarbazine (D group) or carboplatin/paclitaxel (CP group) in patients with previously untreated advanced melanoma. The primary objective was to estimate the effect of ipilimumab on the pharmacokinetics (PK) of dacarbazine and paclitaxel and, conversely, to estimate the effects of dacarbazine and carboplatin/paclitaxel on the PK of ipilimumab. Secondary objectives included evaluation of the safety and anti-tumor activity of ipilimumab when administered alone or with either dacarbazine or carboplatin/paclitaxel, and assessment of pharmacodynamic (PD) effects of ipilimumab on the immune system when administered alone or with either of the two chemotherapies. Ipilimumab was administered at a dose of 10 mg/kg intravenously (IV) every 3 weeks for up to 4 doses. Patients in the D group received dacarbazine 850 mg/m(2) IV every 3 weeks. Patients in the CP group received paclitaxel 175 mg/m(2) IV and carboplatin [AUC=6] IV every 3 weeks. Starting at week 24, patients without dose-limiting toxicities were eligible to receive maintenance ipilimumab at 10 mg/kg every 12 weeks until disease progressed or toxicity required discontinuation. Of 59 randomized patients, 18 (30.5%) discontinued treatment due to adverse events. Response rates by modified WHO criteria were 29.4% (I group), 27.8% (D group), and 11.1% (CP group). No major PK or PD interactions were observed when ipilimumab was administered with dacarbazine or with the carboplatin/paclitaxel combination. This study demonstrated that ipilimumab can be combined safely with two chemotherapy regimens commonly used in advanced melanoma.
Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a key negative regulator of T cell activation. A complex integration of positive and negative co-stimulatory signals in the well-defined B7:CD28/CTLA-4 pathway modulates the generation and maintenance of immune responses. Inhibiting negative regulation through binding of CTLA-4 has been shown to promote stimulation of adaptive immunity and potentiation of T cell activation. CTLA-4-blocking antibodies have demonstrated efficacy in various murine malignancy models when administered as monotherapy; additionally, they have shown synergistic anti-tumor activity when utilized with other agents, such as vaccines, chemotherapy, and radiation. Preclinical studies have supported the rationale for current clinical development of anti-CTLA-4 antibodies, including ipilimumab and tremelimumab, as novel therapeutic strategies to augment anti-tumor immunity in cancer. Both ipilimumab and tremelimumab have been evaluated extensively in melanoma; notably, ipilimumab was recently approved as monotherapy for the treatment of advanced melanoma. Tremelimumab is currently undergoing evaluation in phase II trials as monotherapy in melanoma and malignant mesothelioma, while ipilimumab is under clinical investigation in phase II and III trials in various tumor types, including in melanoma, prostate, and lung cancers as monotherapy and with other therapeutic modalities, such as chemotherapy and radiation. In this review, we will provide a detailed overview of preclinical advances that have delineated many features of CTLA-4 and have helped define its role in T cell response. We will also highlight clinical application of anti-CTLA-4 therapy in cancer and describe knowledge gaps that future studies may address.
Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1-sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination.
The aims of this study were to evaluate the prognostic significance of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) in patients with familial ovarian cancer. Clinical and pathological information were retrieved from the Gilda Radner Familial Ovarian Cancer Registry (GRFOCR) in Buffalo, NY. Immunohistochemistry was performed on paraffin-embedded tissue specimens of GRFOCR participants using specific antibodies for CD3+, CD8+, CD25+, FOXP3+, CD68+, and CD163+. The correlation between the frequencies of TILs and TAMs and clinic-pathologic parameters were determined. Overall survival was determined using univariate and multivariate Cox proportional hazards models. High tumor grade correlated with higher frequencies of CD3+ (p = 0.019), CD68+ (p = 0.025), CD163+ (p = 0.018), and T(reg) (CD25+ FOXP3+) (p = 0.024) cells. Higher stage correlated with higher frequencies of CD163+ cells (p = 0.012). There were correlations between the frequencies of CD68+ and CD3+ (p = 0.029), between T(reg) and each of CD3+ (p = 0.002), CD8+ (p = 0.018), and CD68+ (p = 0.028) cells. In univariate analysis, age and T(reg) significantly predicted patient survival. In multivariate survival analysis, T(reg) frequency was the only significant predictor of prognosis in patients with familial ovarian cancer [HR = 0.92; 95% CI 0.87 - 0.98; p = 0.012]. We concluded that interaction between TILs and TAMs in familial EOC also exists, and tumors with high T(reg) frequencies have a more favorable outcome. Thus, therapeutic strategies to modulate tumor T(reg) infiltration could be beneficial for patients with familial ovarian cancer.
The heat shock proteins (HSPs) gp96 and HSP70 mediate potent antigen-dependent anti-tumor T cell responses in both mammals and Xenopus laevis. We have shown that frogs immunized with total HSP70 generate CD8+ T cell responses against the Xenopus thymic lymphoid tumor 15/0 that expresses several non-classical MHC class Ib (class Ib) genes, but no classical MHC class Ia (class Ia). In the absence of class Ia, we hypothesized that hsp72 can prime class Ib-mediated anti-tumor unconventional CD8+ T cells in an antigen-dependent manner. To test this, we produced Xenopus recombinant HSP70 proteins (both the cognate hsc73 and the inducible hsp72) from stable 15/0 tumor transfectants. We used an in vivo cross-presentation assay to prime animals by adoptive transfer of HSP-pulsed antigen-presenting cells (APCs) and showed that both hsp72-and hsc73-Ag complexes have a similar potential to elicit class Ia-mediated T cell responses against minor histocompatibility (H) Ag skin grafts. In contrast, our in vivo cross-presentation assay revealed that hsp72 was more potent than hsc73 in generating protective immune responses against the class Ia-negative 15/0 tumors in an Ag-dependent and class Ib-mediated manner. These results suggest that hsp72 can stimulate class Ib-mediated immune responses and represents a promising candidate for immunotherapy against malignancies with downregulated class Ia expression.
We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.
Apoptotic cells are significantly more immunogenic than necrotic cells, even though both forms are identical in antigenic content. When a combination of apoptotic and necrotic cells are used to immunize, the phenotype conferred by apoptotic cells, i.e., high immunogenicity, is dominant. However, necrotic cells are not immunosuppressive or tolerogenic. Apoptotic and necrotic cells are taken up by antigen-presenting cells in an equivalent manner. The priming of naïve T cell response is also equivalent. However, the CD8+ T cells elicited by apoptotic cells expand, accumulate, and express effector function, while those primed by the necrotic cells do not. This dichotomy does not extend to CD4+ cells. Apoptotic and necrotic cells elicit equivalent CD4+ T cell priming, accumulation, and function. The deficit in CD8+ T cell function elicited by necrotic cells can be overcome to varying degrees by anti-CD40 antibody and ligands for TLR4 and TLR9; conversely, the immunogenicity of apoptotic cells can be abrogated by blocking anti-CD154 antibody. Our results indicate that immunization with apoptotic cells leads to engagement of CD40 on antigen-presenting cells; this is essential for their ability to elicit mature functional CD8+ cells. The necrotic cells fail to engage CD40, and this failure is the basis of their lack of immunogenicity. These differences have consequences for the understanding of mechanisms of cross-presentation and for efforts toward immunotherapy of cancers and autoimmune pathologies.
Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10-20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI.
When Toshitada Takahashi was in Nagoya, Japan, in 1972, one year after he left New York, he received a phone call from Lloyd Old, asking him to return to the Sloan-Kettering Institute (SKI); Dr. Old wished to organize a human tumor immunology group. By the early 1970s, tumor-associated antigens such as carcinoembryonic antigen (CEA) and α-fetoprotein had been defined by analysis of heteroimmune sera, and the association of Epstein-Barr virus (EBV) with Burkitt’s lymphoma and nasopharyngeal carcinoma had been demonstrated. However, the most important unanswered question was whether tumor cells express antigens that are able to induce host immune responses, ultimately resulting in tumor regression. Around that time, attempts were made by various investigators to define such antigens, predominantly by analyses of the reactions obtained by allogeneic combination of cultured tumor cells and sera and/or lymphocytes from patients. At SKI, the members of the cell-mediated immunity subgroup under Herbert F. Oettgen, Michael A. Bean, and Yoshihisa Kodera were already very actively working on melanoma patients. So, the newly organized virology subgroup of Gaetano Giraldo and our serology subgroup joined together to form the human tumor immunology group headed by Dr. Old, who was just promoted to vice president of Memorial Sloan-Kettering Cancer Center (MSKCC) and associate director of SKI. When Dr. Takahashi met Dr. Old for the first time in his office to initiate a group of human cancer serology in 1973, Dr. Old told him that there was not yet a textbook for human tumor immunology, and that they would be exploring a new research field. There was a concern that analyses based on the allogeneic combination of cultured tumor cells and sera and/or lymphocytes from patients may result in detection of alloantigens, such as blood group antigens and histocompatibility antigens, rather than tumor antigens. Consequently, we decided to apply our approach of autologous typing with serological techniques; in other words, we restricted our analysis to the study of autologous reactions (reaction between sera and tumor cells from the same patient). We primarily chose analyses of serology rather than cell-mediated immunity because of clarity of specificity (1). We also had much experience, in the 1960s, in serology for murine immunogenetic research. For serological techniques, we used rosette assays, such as the immune adherence (IA) assay to detect complement-dependent antibody, predominantly IgM, and mixed hemadsorption assay (MHA) for IgG detection; both techniques are very sensitive and convenient ways to detect surface antigens on cells grown in monolayers, and both are superior to complement-dependent cytotoxicity microassays. The most difficult part of autologous typing was deriving cultures of target tumor cells from solid tumors, but we were very lucky to have Lois A. Resnick working as a technical assistant in our laboratory, a very hard worker with ‘magic hands’ for cell culture. Thanks to the careful arrangement by Dr. Oettgen, we were able to obtain and cultivate various types of tumors, and found that the highest success rate was with melanoma (∼25–30%), with slightly lower success with renal cancer and glioblastoma (∼10–20%). The success rates for other tumors were much lower, and we therefore chose these three tumor types for further study. We conducted autologous typing of sera from 75 melanoma patients. Four patients were found to have antibodies identifying individually distinct tumor antigens (class I), and five patients had antibodies detecting shared tumor antigens (class II). Among class I, the AU antigen, defined by Thomas E. Carey (2), was further studied, since antibody titers residing in the IgG fraction were relatively high, up to 1/256 by MHA; however, characterization of the AU antigen by the conventional radioimmunoprecipitation technique was not possible. Instead, we used antibody inhibition assays to follow antigen solubilization and characterization. AU antigen is easily solubilized by papain and has a molecular weight in the range of 20,000–50,000; unfortunately, the gene encoding for this antigen was not isolated (3). Among class II, the AH antigen was most extensively analyzed. This antigen was defined by Hiroshi Shiku from Nagoya, who joined as a member of the serology group (4, 5), while working with Eiichi Nakayama on Lyt-phenotyping of T cell functions. AH antibody was present in the IgM fraction of the sera of a melanoma patient who had remained alive for six years after resection of recurrent melanoma. This antigen has been found on 70% of melanomas and almost all glioblastomas, but not on normal cultured cells. Biochemical characterization of AH antigen was attempted, but it proved very difficult because of our limited knowledge and experience of membrane chemistry. We asked Kenneth O. Lloyd, from the Texas Tech University School of Medicine in Lubbock, to join as a member of the human tumor immunology group; he kindly accepted our offer in 1976. In 1982, Takeo Watanabe and Kenneth Lloyd demonstrated that the AH antigen molecule resides on a glycolipid molecule, GD2 (6). It is noteworthy in this context that analysis of mouse monoclonal antibodies against melanoma also demonstrated that GD2 and other gangliosides such as GD3 and GM2 are tumor-associated antigens (7). The order of immunogenicity of gangliosides in humans appears to be first GD2, then GM2, and last GD3, whereas in mice, GD3 is the most immunogenic. Later, Philip Livingston and Herbert Oettgen carried out melanoma vaccine programs using gangliosides, such as GM2, as immunizing antigens (8). Massive efforts were made by Hiroshi Yamaguchi and Koichi Furukawa (7, 9) to produce human monoclonal antibodies from immunized and non-immunized patients; two such antibodies were identified: one detecting GM3, and the other detecting GD3. Both antibodies exhibited selective reactivity with tumor cells of neuroectodermal origin, particularly melanomas. As for the biochemical characterization of unique antigens, the first report by Drs. Furukawa and Lloyd appeared in 1989; that study demonstrated that a unique FD antigen epitope is carried on the common melanoma glycoprotein gp95/p97 (also known as melanotransferin) (10). In addition to melanoma, autologous typing of glioblastoma and renal cancer was conducted by Michael Pfreundschuh (11) and Ryuzo Ueda (12), respectively. In the case of glioblastoma, sera from two patients (from a total of 30 tested) recognized class I antigens, while four recognized class II antigens. Interestingly, one of the class II antigens was found to be serologically related to the AH antigen of melanoma, i.e., GD2 antigen. In Ueda’s study of 28 patients with renal cancer, sera from one patient detected a class I antigen, and sera from three recognized class II antigens. Autologous typing of cryopreserved leukemia cells was also conducted by Thomas J. Garrett (13); sera from one patient detected a common acute lymphocytic leukemia (ALL) antigen. A similar study was conducted later in Japan by Kazuyuki Naito (14), who also reported a common ALL antigen. Thus, these autologous typing studies clearly indicated the presence of tumor antigens that are able to induce antibody responses in patients. It is noteworthy that this approach laid the foundation for the development of a novel serological approach, SEREX (serological identification of antigens by recombinant expression cloning), by Dr. Pfreundschuh and his colleagues, Ugur Sahin and Ozlem Tureci, in 1995 (15). SEREX enables definition of tumor antigens from any tumor type. Autologous typing was also applied to T cell-mediated immunity by Alexander Knuth (16), leading to the establishment of killer T cell lines that allowed the isolation of tumor-specific antigen genes. More detailed stories about how autologous typing helped lay the groundwork for new methods to identify tumor antigens recognized by antibodies and T cells will be told later in this issue by Drs. Pfreundschuh and Knuth, respectively.
Although Lloyd J. Old was involved in various studies of the interactions between cancer and the immune system, it seems to us that his ideas often originated from the studies of serological identification of mouse lymphocyte antigens. The findings from these studies allow us to distinguish cells of different lineages and differentiation stages, and also to distinguish leukemia cells from normal lymphocytes (1). It is amazing that essentially a single serological technique, i.e., the Trypan blue exclusion test (presumably introduced in Peter Gorer’s laboratory by Edward A. Boyse), was used to define a series of these antigens. This technique was used in conjunction with absorption analysis to elucidate the specificity of antisera, based on a vast knowledge of mouse immunogenetics. In the laboratory, Elisabeth Stockert (who was a technical assistant at that time) was the great master of these serological techniques, carrying out her own projects of cell surface antigens, as will be described below, while managing the day-to-day business of the laboratory. Similarly, Elizabeth A. Carswell and Gayla Geering investigated tumor necrosis factor (TNF) and retrovirus-associated intracellular antigens, respectively, while working as technical assistants.
Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10-20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI.
