Integrating genomics and proteomics permits identification of immunodominant antigens associated with drug resistance in human visceral leishmaniasis in India.

Resistance of human pathogens like Leishmania to drugs is a growing concern where the multidrug-resistant phenotype renders chemotherapy ineffective. The acquired resistance of Leishmania to antimony has promoted intense research on the mechanisms involved but the question has not been resolved yet. In this study we have explored host-pathogen- drug interactions leading to identification of pharmacological determinants of host macrophages that resist the sodium antimony gluconate (SAG) mediated intracellular parasite killing. mRNA profiling of mammalian host stage amastigotes of sodium antimony gluconate (SAG) 'sensitive' and 'resistant' parasite lines was carried out using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Patient sera was used to identify immunogenic proteins by two-dimensional gel analysis (2DE) and mass spectrometric analysis (LC-MS/MS). Immunofluorescence microscopy confirmed the identities on 'sensitive' and 'resistant' parasite lines. A total of nine immunogenic proteins whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins are altered in expression levels in the 'resistant' parasites. Global expression profiling using microarrays revealed this regulation was not reflected by changes in the levels of the cognate mRNAs. Following identification of proteins by mass spectrometry, one such regulated protein, enolase, was chosen for more detailed analysis. Immunofluorescence microscopy employing antisera against this enzyme confirmed that its level was differentially regulated in the 'resistant' isolate. We show that high serum level of immunoreactive protein is associated with 'resistant' phenotype. Differentially expressed proteins with immunomodulatory activities were found to be associated with the 'resistant phenotype'.

Clinical utility of myb rearrangement detection and p63/p40 immunophenotyping in the diagnosis of adenoid cystic carcinoma of minor salivary glands: a pilot study.

OBJECTIVES: MYB rearrangement is observed in approximately 28% to 86% of adenoid cystic carcinomas (ACCs). Also, ACC features a p63+/p40+ immunophenotype in greater than 90% of cases, compared with p63+/p40- polymorphous low-grade adenocarcinoma (PLGA). Our aim was to investigate the incidence of (1) MYB rearrangement and (2) p63/p40 immunoreactivity in ACC and PLGA of minor salivary glands (MSGs). STUDY DESIGN: Seven cases of ACC as well as five of PLGA were evaluated by using a MYB (6 q23.3) break-apart fluorescence in situ hybridization (FISH) probe. In addition, all cases were immunohistochemically stained with p63 and p40 antibodies. RESULTS: All five successfully hybridized ACCs featured MYB rearrangement, whereas PLGAs did not show MYB rearrangement. Interestingly, one case of PLGA demonstrated a single intact copy of MYB in greater than 88% of the neoplastic cells. All ACCs exhibited consistent p63+/p40+ staining, whereas PLGAs demonstrated a p63+/p40- immunophenotype. CONCLUSIONS: (1) MYB rearrangement is encountered in ACCs but not PLGAs of MSGs; (2) MYB aberrations, for example, monosomy or deletion, can be seen in PLGAs; (3) combined p63/p40 immunostaining can be used to differentiate ACC from PLGA in incisionally biopsied specimens; and (4) performance of either FISH or p63/p40 immunohistochemistry is expected to be able to confirm the diagnosis of ACC or PLGA in small intraoral biopsies, since both techniques appeared to be diagnostically accurate in this pilot study.

Desmoglein 3 and p40 immunoreactivity in neoplastic and nonneoplastic thymus: a potential adjunct to help resolve selected diagnostic and staging problems.

The potential usefulness of the squamous markers p40 and desmoglein 3 (DSG-3) for the diagnosis and staging of selected thymic lesions is uncertain. We investigated their expression and distribution pattern in 66 thymomas, 12 thymic squamous carcinomas, 6 undifferentiated thymic carcinomas, 5 hyperplastic thymi, and 5 normal thymi. p40 nuclear and DSG-3 cytoplasmic/membranous immunoreactivity in greater than or equal to 10% of thymic epithelial cells was interpreted as positive, and DSG-3 distribution pattern was classified as organotypic and nonorganotypic. All nonneoplastic thymic tissues, 100% of thymic squamous carcinomas, 97% of thymomas, and 50% of undifferentiated thymic carcinomas were positive for p40. Expression of p40 in almost all thymomas and in 50% of undifferentiated carcinomas that lacked squamous features suggests that p40 is not a good marker for the diagnosis of thymic squamous carcinoma. All normal and hyperplastic thymi, 51.5% of thymomas, and 0% of thymic squamous carcinomas expressed DSG-3 in an organotypic pattern, and 13.6% of thymomas and 83% of thymic squamous carcinomas were DSG-3 positive in a nonorganotypic pattern. Findings suggest that nonorganotypic DSG-3 expression favors the diagnosis of squamous cell carcinoma over thymoma. In 26 (60.5%) of the 43 cases where neoplastic and nonneoplastic thymus were present on the same slide, the presence/absence or distribution pattern of DSG-3 immunoreactivity was different in the 2 components, suggesting that this marker can be helpful in staging thymomas with incomplete encapsulation. The presence of DSG-3-positive and DSG-3-negative thymomas raises the possibility that these tumors may originate from 2 different types of thymic epithelial cells.

Primary pulmonary hyalinizing clear cell carcinoma of bronchial submucosal gland origin.

Hyalinizing clear cell carcinoma (HCCC) has only been described in salivary glands of the head and neck. We report a 38-year-old man with a 2.6-cm lung tumor that was growing in a peribronchial location and had morphologic features of HCCC. The tumor cells expressed cytokeratin 7 and keratin AE1/AE3, and the vast majority of tumor cells marked also with p63 and p40. They were negative for cytokeratin 20, S-100, smooth muscle actin, napsin A, and thyroid transcription factor-1. Fluorescence in situ hybridization revealed Ewing Sarcoma Breakpoint Region 1 (EWSR1) rearrangement, and reverse-transcription polymerase chain reaction confirmed the presence of the EWSR1-Activating Transcription Factor 1 (ATF1) fusion transcript, which was subsequently sequenced. The morphologic, immunophenotypic, cytogenetic, and molecular findings together with the patient's history and location of the tumor support a diagnosis of primary pulmonary HCCC of bronchial submucosal gland origin. It is our understanding that this is the first report of HCCC arising as a primary tumor outside the head and neck region.

p40 is more specific than p63 for the distinction of atypical fibroxanthoma from other cutaneous spindle cell malignancies.

Poorly differentiated, cytologically malignant, spindle cell neoplasms of the skin may present a diagnostic challenge with important clinical consequences. In particular, the distinction between poorly differentiated cutaneous spindle cell squamous cell carcinoma (SpSCC) and atypical fibroxanthoma (AFX) remains controversial, but with important clinical implications: SpSCC exhibits an increased tendency for both local recurrence and metastasis compared with AFX. AFX is generally accepted as a diagnosis of exclusion based on negativity for a broad panel of immunohistochemical markers, including multiple cytokeratins, melanocytic markers, muscle markers, and vascular markers. As cytokeratins can also be occasionally lost in SpSCC, it would be of tremendous diagnostic value if there were additional specific markers to facilitate the distinction of lineage in this differential diagnostic context. Initial studies demonstrated p63 to be of utility in distinguishing AFX from SpSCC; however, p63 has proved to lack specificity, as it also exhibits variable reactivity in a subset of AFX. Recent studies have shown p40 immunohistochemistry to be a more specific marker than p63 for the designation of squamous differentiation in carcinomas involving other organ systems. In the current study, we define the utility of p40 immunohistochemistry among common cutaneous spindle cell malignancies, and, specifically, we compare the diagnostic accuracy of p40 and p63 in distinguishing AFX from SpSCC. We show that p40 and p63 exhibit comparable sensitivity, but p40 exhibits superior specificity in the distinction of AFX from SpSCC.

The utility of a novel triple marker (combination of TTF1, napsin A, and p40) in the subclassification of non-small cell lung cancer.

In lung cancer, targeted therapies depend on accurate histological subclassification of the tumor. The majority of lung cancers can be subclassified based on hematoxylin and eosin staining; however, classification may be difficult in small biopsies. In this study, we investigated the utility of a newly developed triple marker (combination of TTF1/Napsin A/p40) and compared the sensitivity and specificity of this novel marker with individual markers in the subclassification of non-small cell lung carcinomas. Lung cancer tissue microarrays were constructed using surgical resection material from the Johns Hopkins Hospital. They included 77 adenocarcinomas (ADCs), 77 squamous cell carcinomas (SqCCs), and 46 cases of metastatic lung ADCs. Immunostaining patterns of all markers were scored semi-quantitatively and compared. In ADCs, the sensitivity and specificity of the triple marker were 93.5% and 77.5%, respectively. The sensitivity and specificity of TTF1 and Napsin A were 85.7% and 75.0%, and 89.6% and 90.0%. In SqCCs, the sensitivity and specificity of the triple marker were 88.3% and 92.5%, while the p40, p63 and CK5/6 showed 80.5% and 90.0%; 93.5% and 80.0%; and 89.6% and 80.0%. In addition, the sensitivity and specificity of the triple marker in metastatic ADCs showed 71.7% and 73.5%, respectively. Our triple marker (combination of TTF1/Napsin A/p40) showed a similar sensitivity and specificity for the subclassification of NSCLC when compared to individual markers. Our study not only demonstrates a useful combination of immunomarkers but also optimally conserves tissue for molecular marker testing.

T cell epitope mapping of JC polyoma virus-encoded proteome reveals reduced T cell responses in HLA-DRB1*04:01+ donors.

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4(+) T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4(+) T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01(+), DRB1*07(+), DRB1*11(+), DRB1*13(+), DRB1*15(+), and DRB1*03(+) individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01(+) individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.

Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies.

Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.

Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.

Identification of carbonic anhydrase I immunodominant epitopes recognized by specific autoantibodies which indicate an improved prognosis in patients with malignancy after autologous stem cell transplantation.

This work employs an epitope mapping of carbonic anhydrase (CA), isoform I (CA I), for detection of the main immunodominant epitopes. Our interest has arisen from an observed spontaneous tumor regression in patients who developed an aplastic anemia type syndrome after a high-dose therapy with autologous stem cell transplantation and whose sera contained high titer of anti carbonic anhydrase (anti-CA) autoantibodies. There are many indications that the presence of these autoantibodies may provide significant survival benefit for the patients. Western blot analysis confirmed strong immunoreactivity of the patients' sera with several CA isoforms and the CA I has been selected for our study as a highly abundant and widely distributed isoform. The applied analytical approach consists of specific fragmentation of CA I protein followed by immunospecific isolation of peptides reacting with polyclonal anti-CA I autoantibodies of patients in spontaneous remission. We improved the standard epitope mapping schema by incorporating the benefits of magnetic carriers and biomagnetic separation techniques. Mass spectrometry has been applied for detection and identification of epitopes and the acquired results were verified by bioinformatic tools. The candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) are discussed herein as potential therapeutic targets. This work highlights the usefulness of the epitope mapping technique based on magnetic microspheres for effective and rapid determination of immunodominant epitopes of the target protein.

QUASI analysis of host immune responses to Gag polyproteins of human immunodeficiency virus type 1 by a systematic bioinformatics approach.

There is a consensus that Gag-specific cytotoxic T lymphocyte (CTL) response plays a key role in the immune control of human immunodeficiency virus type 1 (HIV-1) infection. In this study, we analyzed all currently available gag sequences in the Los Alamos HIV sequence database and identified positive selection (PS) sites likely restricted by the host immune responses. We found that between 23.4% and 47.4% of PS sites were shared by clades A, B, and C of Gag, indicating similar positive selection pressure on Gag in different subtypes of HIV-1. Furthermore, a significant correlation was observed between the combined CTL and antibody responses and PS sites. The Gag regions of free from PS contained 9 CTL epitopes restricted by 11 HLA class I alleles associated with disease progression to acquired immune deficiency syndrome (AIDS). These analyses provide information important for the identification of cross-clade epitopes and development of a global HIV-1 vaccine.

Serologic markers of effective tumor immunity against chronic lymphocytic leukemia include nonmutated B-cell antigens.

Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens-C6orf130, MDS032, and ZFYVE19-were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity.

A degenerate HLA-DR epitope pool of HER-2/neu reveals a novel in vivo immunodominant epitope, HER-2/neu88-102.

PURPOSE: Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell-activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu-specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas. EXPERIMENTAL DESIGN: A combination of algorithms and HLA-DR-binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-gamma enzyme-linked immunosorbent spot (ELIspot) assay. RESULTS: Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for > or =11 common HLA-DR molecules. A degenerate pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu(88-102) (p88). CONCLUSION: The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu-targeting, immune-based therapies such as vaccines.

Identification of epitopes and immunodominant regions on the MICA protein defined by alloantibodies from kidney transplant patients.

BACKGROUND: Several reports showed a contribution of anti-MICA (major histocompatibility complex class I chain-related molecule A) antibodies (Abs) to the development of acute and chronic rejection. Identification of the epitopes to which the Abs bind may help to determine immunoreactive regions essential for the major histocompatibility complex compatibility between donor and recipients, leading to the best outcome of the transplant. METHODS: Sera from 284 kidney transplant patients were screened for anti-MICA Abs by Luminex assay. MICA allele typing of the recipients was determined. The epitopes of MICA were mapped by screening a synthesized library of overlapping peptides from the extracellular domains of the protein against the sera from kidney transplant patients with anti-MICA Abs. RESULTS: Antibodies against MICA were detected in 50 of 284 patients (17.6%) and correlated with the development of acute rejection. Nine antigenic regions were immunoreactive with anti-MICA Abs in the sera samples. Four of these continuous epitopes mapped to polymorphic amino acids (aa). Five antigenic regions were shared epitopes found in all the MICA alleles. The polymorphic residues, 173 (E/K), 175 (S/G), and 181 (R/T), had determined allele-specific epitopes (reactivity patterns 1 and 2). In contrast, the aa 208Y and 213T were implicated in the cross-reactivity among alleles. CONCLUSIONS: The presence of anti-MICA Abs could be an important marker for diagnosis because of their contribution to the outcome of the graft, regardless of presence of anti-HLA Abs. Additionally, the identification of epitopes revealed the in vivo antigens of the transplant and is spurring the development of new matching strategies to reduce the incidence of acute and chronic rejection.

Vaccination with recombinant NY-ESO-1 protein elicits immunodominant HLA-DR52b-restricted CD4+ T cell responses with a conserved T cell receptor repertoire.

PURPOSE: ESO is a tumor-specific antigen with wide expression in human tumors of different histologic types and remarkable spontaneous immunogenicity. We have previously shown that specific T(H)1 and antibody responses can be elicited in patients with no detectable preexisting immune responses by vaccination with rESO administered with Montanide ISA-51 and CpG ODN 7909. The purpose of the present study was to characterize vaccine-induced ESO-specific CD4(+) T cell responses. EXPERIMENTAL DESIGN: We generated CD4(+) T cell clones from patient C2, who had the highest CD4(+) T cell response to the vaccine, and analyzed their fine specificity and HLA class II restriction to determine the recognized epitope. We then assessed the response to the identified epitope in all vaccinated patients expressing the corresponding HLA class II allele. RESULTS: We found that ESO-specific CD4(+) T cell clones from patient C2 recognize peptide ESO(119-143) (core region 123-137) presented by HLA-DR52b (HLA-DRB3*0202), a MHC class II allele expressed by about half of Caucasians. Importantly, following vaccination, all patients expressing DR52b developed significant responses to the identified epitope, accounting for, on average, half of the total CD4(+) T cell responses to the 119-143 immunodominant region. In addition, analysis of ESO-specific DR52b-restricted CD4(+) T cells at the clonal level revealed significant conservation of T cell receptor usage among different individuals. CONCLUSIONS: The identification of a DR52b-restricted epitope from ESO that is immunodominant in the context of vaccine-elicited immune responses is instrumental for the immunologic monitoring of vaccination trials targeting this important tumor antigen.

Enhanced antigen presentation and CTL activity by transduction of mature rather than immature dendritic cells with octaarginine-modified liposomes.

To improve uptake and cross-presentation of exogenous antigens (Ag) by dendritic cells (DCs), octaarginine-modified liposomes (R8-Lip) were used as a novel strategy for protein-Ag transduction. Immature DCs endocytose macromolecules efficiently. While mature DCs lose their ability to capture Ag, but have an increased capacity for T-cell activation. Thus Ag-transduction has been performed mostly in immature DCs. In the present study, R8-Lip were efficiently taken up by both immature and mature DCs. DCs transduced after maturation were highly efficient at cross-presentation of Ag and induced higher cytotoxic T-lymphocytes (CTL) activity than were DCs transduced before maturation. The mechanism of Ag presentation involved the escape of R8-Lip from endosomes to cytosol, which require the acidic environment. The Ag released was then processed by a proteasome-dependent pathway. This novel transduction approach is clinically applicable, easy to perform, and has more practical advantages than current protein transduction methods.

Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines.

Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most-if not the most-frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy.