Thomas Wölfel

A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma

A mutated cyclin-dependent kinase 4 (CDK4) was identified as a tumor-specific antigen recognized by HLA-A2. 1-restricted autologous cytolytic T lymphocytes (CTLs) in a human melanoma. The mutated CDK4 allele was present in autologous cultured melanoma cells and metastasis tissue, but not in the patients lymphocytes. The mutation, an arginine-to-cysteine exchange at residue 24, was part of the CDK4 peptide recognized by CTLs and prevented binding of the CDK4 inhibitor p16INK4a, but not of p21 or of p27KIP1. The same mutation was found in one additional melanoma among 28 melanomas analyzed. These results suggest that mutation of CDK4 can create a tumor-specific antigen and can disrupt the cell-cycle regulation exerted by the tumor suppressor p16INK4a.

Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation

Adoptive cell transfer therapies (ACTs) with cytotoxic T cells that target melanocytic antigens can achieve remissions in patients with metastatic melanomas, but tumours frequently relapse. Hypotheses explaining the acquired resistance to ACTs include the selection of antigen-deficient tumour cell variants and the induction of T-cell tolerance. However, the lack of appropriate experimental melanoma models has so far impeded clear insights into the underlying mechanisms. Here we establish an effective ACT protocol in a genetically engineered mouse melanoma model that recapitulates tumour regression, remission and relapse as seen in patients. We report the unexpected observation that melanomas acquire ACT resistance through an inflammation-induced reversible loss of melanocytic antigens. In serial transplantation experiments, melanoma cells switch between a differentiated and a dedifferentiated phenotype in response to T-cell-driven inflammatory stimuli. We identified the proinflammatory cytokine tumour necrosis factor (TNF)-α as a crucial factor that directly caused reversible dedifferentiation of mouse and human melanoma cells. Tumour cells exposed to TNF-α were poorly recognized by T cells specific for melanocytic antigens, whereas recognition by T cells specific for non-melanocytic antigens was unaffected or even increased. Our results demonstrate that the phenotypic plasticity of melanoma cells in an inflammatory microenvironment contributes to tumour relapse after initially successful T-cell immunotherapy. On the basis of our work, we propose that future ACT protocols should simultaneously target melanocytic and non-melanocytic antigens to ensure broad recognition of both differentiated and dedifferentiated melanoma cells, and include strategies to sustain T-cell effector functions by blocking immune-inhibitory mechanisms in the tumour microenvironment.

Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor α in response to HLA-A2.1-binding melanoma and viral peptide antigens

We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.

The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor alpha spots in response to peptide antigens.

Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor alpha (TNF-alpha) spots formed by single blood-derived CD8+ T cells after contact with peptide-loaded target cells. With CVIA and TNF-alpha ELISPOT analysis we quantified CD8+ T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reactivation of latent cytomegalovirus (CMV) infection during immunosuppressive therapy. The test proved sufficiently sensitive to detect in the blood of both patients a temporary expansion of CD8+ T lymphocytes reactive with a known immunogenic HLA-A2.1-binding peptide from glycoprotein B of CMV. Reactivity to peptide antigens was not only reflected by numeric increases of spot formation, but also by the appearance of larger spot areas, presumably formed by strongly peptide-reactive CD8+ T cells. We conclude that the combined use of the TNF-alpha ELISPOT assay and CVIA allows reliable monitoring of the T cell responsiveness to peptide antigens in peripheral blood.

The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8+ T lymphocytes in IFN-γ ELISPOT assays

ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to strong background spot production by APC-reactive T lymphocytes or have a low antigen presentation capability. Both phenomena can prevent the detection of low frequency T cell responses in PBMC. In search of alternative APC for ELISPOT assays, the human chronic myelogenous leukemia cell line K562 that per se does not express HLA class I and class II molecules on the cell surface was transfected with the HLA-A*0201 gene. Clonal HLA-A*0201-expressing K562 (K562/A*0201) cells were able to process and present endogenously expressed and exogenously loaded melanoma peptide antigens to HLA-A*0201-restricted cytolytic T lymphocyte clones in cytotoxicity and IFN-gamma ELISPOT assays. K562/A*0201 cells were then used as APC in IFN-gamma spot assays to detect ex vivo CD8(+) T lymphocytes responsive to known HLA-A*0201-binding peptide epitopes derived from cytomegalovirus, Epstein-Barr virus, influenza virus and melanoma in PBMC from HLA-A*0201-positive donors. In the majority of cases, peptide-pulsed K562/A*0201 cells were similarly efficient in the ability to visualize single antigen-specific CD8(+) T lymphocytes when compared to T2 cells. However, in contrast to T2, background reactivity of CD8(+) T cells responsive to unpulsed K562/A*0201 was regularly found to be negligible, thereby enhancing the sensitivity of the ELISPOT assay, particularly in donors with strong anti-T2 reactivity. K562 cells transfected with HLA-A*0201 or other HLA genes can serve as standard APC for monitoring T lymphocyte responses against tumor and viral peptide antigens.

Overlapping peptides of melanocyte differentiation antigen Melan-A/MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA-A2.1.

From the peripheral blood lymphocytes (PBLs) of melanoma patient SK29(AV) we have previously isolated 2 independent cytolytic T lymphocyte (CTL) clones (CTL7/147 and CTL13/211), which lysed autologous tumor cells in association with HLA‐B45.1. As demonstrated here, both CTL clones were directed against melanocyte differentiation antigen Melan‐A/MART‐1, which also was recognized by HLA‐A2.1‐restricted CTLs from the same patient. By generating and transfecting 3′‐deletion mutants of Melan‐A/MART‐1 cDNA, we localized its peptide‐coding regions. The HLA‐B45.1‐presented peptides were derived from a hydrophobic region of the protein and largely overlapped the peptides recognized by CTLs from the same patient in association with HLA‐A2.1. We determined the fine specificity of these CTL clones with synthetic peptides. CTL clone CTL7/147 recognized the 11‐mer peptide AEEAAGIGILT (residues 24–34) at the lowest concentrations. The absence of threonine‐34 abrogated the recognition by CTL7/147. The truncated peptide AEEAAGIGIL (residues 24–33) proved to be the optimal synthetic peptide for sensitization against lysis by CTL13/211. This indicated that C‐terminal threonine‐34 was not involved in binding to HLA‐B45.1 but, rather, was part of the epitope for CTL7/147. HLA‐B45.1‐associated peptides of Melan‐A/MART‐1 were regularly processed and presented by other melanomas and other cell types. Three of 4 independent HLA‐A2.1‐restricted SK29‐CTL clones recognized the 10‐mer peptide EAAGIGILTV (residues 26–35) at 10‐ to 100‐fold lower concentrations than the nonamer AAGIGILTV (residues 27–35), previously described as the common immunodominant peptide antigen for all known anti‐Melan‐A/MART‐1 CTLs restricted by HLA‐A2.1. Different melanoma peptide antigens currently are applied in therapeutic vaccination studies. Our findings emphasize that restricting to peptides of minimal length might exclude relevant T‐cell epitopes. Int. J. Cancer 75:451–458, 1998.

Genes coding for tumor antigens recognized by human cytolytic T lymphocytes.

In order to define the antigens recognized by cytolytic T lymphocytes (CTLs) on autologous tumors, we derived tumor-specific CTL clones from autologous mixed lymphocyte tumor cell cultures. The gene coding for a tumor rejection antigen expressed on a melanoma was isolated by transfecting genomic DNA of the tumor into an antigen-loss variant of the melanoma. Transfectants were identified on the basis of their ability to stimulate tumor necrosis factor release by the CTL clone. The gene that transferred the expression of the antigen was named MAGE-1. It is a new gene, silent in normal tissues with the exception of testis, but expressed in several types of tumors. The antigen recognized by the CTL clone is a nonapeptide derived from the protein encoded by gene MAGE-1, and presented by the HLA class I molecule HLA-A1. Using two other antimelanoma CTL clones, we identified the tyrosinase gene as coding for an antigen presented by HLA-A2 on this type of tumors. The identification of these tumor rejection antigens open new possibilities for the specific immunotherapy of cancer.

A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr)

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5×108 IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I–restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.

TRANSPORTER (TAP)- AND PROTEASOME-INDEPENDENT PRESENTATION OF A MELANOMA-ASSOCIATED TYROSINASE EPITOPE

The melanosomal protein tyrosinase is considered as a target of specific immunotherapy against melanoma. Two tyrosinase‐derived peptides are presented in association with HLA‐A2.1 [Wölfel et al., Eur. J. Immunol., 24, 759–764 ( 1994 )]. Peptide 1‐9 (MLLAVLYCL) is generated from the putative signal sequence. The internal peptide 369‐377 is posttranslationally converted at residue 371, and its presentation is dependent on functional TAP transporters and proteasomes [Mosse et al., J. exp. Med.187, 37–48 ( 1998 )]. Herein, we report on the processing and transport requirements for the signal sequence‐derived peptide 1‐9 that were studied in parallel to those for peptide 369‐377. After infection of TAP‐deficient (T2) and TAP‐positive (T1) cells with a Modified Vaccinia Ankara construct carrying the human tyrosinase gene (MVA‐hTyr), we found that recognition by CTL against peptide 1‐9 did not require TAP function as opposed to recognition by CTL against peptide 369‐377. When target cells with intact processing and transport functions were infected with MVA‐hTyr, lysis by CTL against peptide 1‐9 was not impaired by lactacystin, a specific inhibitor for the proteasome, whereas lysis by CTL against peptide 369‐377 was completely abrogated. Taken together, peptide 1‐9 derived from the signal sequence of tyrosinase is presented in a TAP‐independent fashion and does not require proteasomes for processing. Cellular immune responses against this hydrophobic peptide can be monitored with lymphokine spot assays as documented in the case of a patient with metastatic melanoma, in whom we observed a preferential T‐cell response against tyrosinase peptide 1‐9 subsequent to chemoimmunotherapy. Independence of cytosolic processing and transport pathways and potentially enhanced expression levels make signal sequence‐derived peptides and their carrier proteins important candidates for specific immunotherapy.Int. J. Cancer 88:432–438, 2000.

Quantification of CD8+ T Lymphocytes Responsive to Human Immunodeficiency Virus (HIV) Peptide Antigens in HIV-Infected Patients and Seronegative Persons at High Risk for Recent HIV Exposure

The combination of a tumor necrosis factor (TNF)-alpha enzyme-linked immunospot (ELISPOT) assay and computer-assisted video image analysis was used to detect and quantitate in peripheral blood CD8+ T cells reactive with known human immunodeficiency virus type 1 (HIV-1) peptide antigens presented by HLA-A2 or HLA-A3. T lymphocyte responsiveness to at least one HIV peptide was found in 10 (83%) of 12 HIV-1-infected patients and in 5 (45%) of 11 persons who had no serologic and virologic signs of HIV infection but who were at high risk for recent sexual exposure to HIV-1. CD8+ T cells responding to HIV-1 peptides were observed in none of 11 HIV-seronegative donors without a history of HIV exposure. ELISPOT assays are relatively fast and easy to perform and appear to reliably detect T cell reactivity due to previous exposure to HIV. These findings support the use of the ELISPOT assay for monitoring T cell responsiveness to HIV peptides.