Analysis of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-reactive CD8(+) T cell responses in children with NPM-ALK(+) anaplastic large cell lymphoma.

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

The programmed death (PD)-1/PD-ligand 1 pathway regulates graft-versus-host-reactive CD8 T cells after liver transplantation.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after solid-organ transplantation, which is mediated by host-reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow-infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host-versus-donor reactivity was selectively impaired, as anti-third-party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor-specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3-positive T cells. In fact, graft-versus-host-reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)-1 receptor and its ligand PD-L1. We found high PD-1 expression on donor CD4 and CD8 T cells. In addition, blocking PD-L1 on host-derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD-1/PD-L1 pathway as a therapeutic strategy to control graft-versus-host-reactive T cells in allograft recipients.

Identification of NM23-H2 as a tumour-associated antigen in chronic myeloid leukaemia.

Therapeutic effects of haematopoietic stem cell transplantation are not limited to maximal chemoradiotherapy and subsequent bone marrow regeneration, but include specific as well as unspecific immune reactions known as graft-versus-leukaemia (GvL) effects. Specific immune reactions are likely to be particularly relevant to the long-term treatment of diseases, such as chronic myeloid leukaemia (CML), in which residual cells may remain quiescent and unresponsive to cytotoxic and molecular therapies for long periods of time. Specific GvL effects result from the expression on leukaemic cells of specific tumour-associated antigens (TAAs) in the context of HLA proteins. As human leukocyte antigen (HLA) types vary widely, the development of broadly applicable tumour vaccines will require the identification of multiple TAAs active in different HLA backgrounds. Here, we describe the identification of NM23-H2 as a novel HLA-A32-restricted TAA of CML cells and demonstrate the presence of specifically reactive T cells in a patient 5 years after transplantation. As the NM23 proteins are aberrantly expressed in a range of different tumours, our findings suggest potential applications beyond CML and provide a new avenue of investigation into the molecular mechanisms underlying CML.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays.

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Generating p53-specific cytotoxic T lymphocytes by recombinant adenoviral vector-based vaccination in mice, but not man.

Mutations and aberrant expression of the p53 tumor suppressor protein are the most frequent molecular alterations in human malignancy. Peptides derived from the wild-type (wt) p53 protein and presented by major histocompatibility complex (MHC) molecules for T lymphocyte recognition are believed to serve as universal tumor-associated antigens for cancer immunotherapy. We studied the immunogeneicity of a recombinant replication-defective adenoviral vector encoding human full-length wt p53 (rAd/hup53) in human leukocyte antigen (HLA)-A2K(b)-transgenic (Tg) mice and man. The generation of p53 epitope-specific cytotoxic T lymphocytes (CTLs) in p53-proficient and p53-deficient A2K(b)-Tg mice was affected by self-tolerance and a selective inability of rAd/hup53 to induce p53.264-272 peptide-reactive effector cells. To extend this study into a pilot clinical trial, six advanced-stage cancer patients received sequential injections of rAd/hup53. The treatment was well tolerated. To date, no evidence for objective tumor responses was observed. An amplification of humoral and cellular anti-adenoviral immune responses was demonstrated in all patients following rAd/hup53 vaccination. However, p53-reactive antibodies and HLA-A*0201 (A2.1)-restricted CTLs specific for wt p53 epitopes were not generated. Tailoring p53-based cancer immunotherapy thus requires the interference with p53-specific self-tolerance and the induction of the entire repertoire of p53-reactive T lymphocytes.

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.

Consequences of antigen self-presentation by tumor-specific cytotoxic T cells.

CD8-positive cytotoxic T cells (CTL) recognize antigenic peptides in combination with major histocompatibility complex (MHC) class I molecules on the surface of syngeneic antigen presenting cells (APC). In the present paper we show that cells from tumor antigen-specific CTL clones present their cognate antigenic peptide to other CTL from the same clone. Inter-CTL peptide presentation resulted in activation of the cells of one CTL clone to MHC-unrestricted lysis of bystander cells. In contrast to the behaviour of this clone, another CTL clone did not lyse bystander cells after incubation with the cognate peptide, but was activated to self-destruction. The human herpes virus Epstein-Barr virus is involved in the pathogenesis of a broad spectrum of human neoplasias. Using freshly established non-clonal T cells with specificity for a peptide derived from an Epstein-Barr virus encoded antigen we found again lysis of MHC mismatched bystander cells as a consequence of inter-CTL peptide presentation, indicating that bystander lysis following antigen self-presentation is not a phenomenon restricted to long-term in vitro cultured T cell clones. The potential implications for immunosurveillance against cancer and for tumor escape mechanisms are discussed.

A tyrosinase peptide presented by HLA-B35 is recognized on a human melanoma by autologous cytotoxic T lymphocytes.

We previously described different cytotoxic T lymphocyte (CTL) clones isolated from the blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. These CTL clones recognized at least 2 distinct antigens on the melanoma cells. Here, we show that one of them consists of a peptide derived from tyrosinase and presented by HLA-B35. The peptide is 9 amino acids long and has the sequence LPSSADVEF. It can be presented by the 2 major B35 allelic subtypes, B*3501 and B*3503. As HLA-B35 is one of the most frequent HLA-B specificities, being present in about 20% of Caucasian individuals, it may be a useful target for peptide-based immunotherapy of melanoma.

Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen: induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo.

Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.

[Decreased rate of progression and induction of tumor-specific immune response by adjuvant immunotherapy in stage IV melanoma]. / Verminderte Progressionsrate und Induktion tumorspezifischer Immunantworten durch adjuvante Immuntherapie bei Stadium-IV-Melanomen.

Stage IV melanoma is still a disease with poor prognosis. Although modern chemo- or chemoimmunotherapies give high response rates in stage IV patients, remissions are usually followed by fast relapses. In order to avoid early relapses after chemotherapy, patients with stage IV disease and either stable disease or partial or complete remission following therapy were treated with 9 million IU IFN alpha subcutaneously 5 times weekly and 6 million IU IL-2 subcutaneously twice weekly. Compared with untreated controls, the rate of progression in the treatment group was reduced from 95% to 35%. Also, time to progression was significantly prolonged. Median survival times in the control group were 25 weeks, whereas median survival time in the treatment group has not yet been reached. Furthermore, TNF-ELISPOT assays showed a significant increase in MAGE-3 reactive cytotoxic T-cells in the treatment, but not in the control group. Thus, immunotherapy in stage IV disease seems to prolong survival in melanoma patients.

Quantitative and functional analysis of core-specific T-helper cell and CTL activities in acute and chronic hepatitis B.

AIMS/BACKGROUND: CD4+ T-helper cell (Th) responses to hepatitis B virus (HBV) core antigen (HBc) are increased during exacerbations in acute and chronic hepatitis B (AHB, CHB) and might influence the induction of CD8+ cytotoxic T lymphocytes (CTL) that are important for viral clearance. METHODS: HBc-specific proliferative responses and cytokine release of blood mononuclear cells (PBMC) were studied in patients with AHB or CHB, as well as responders and non-responders to interferon-alpha treatment (IFN-R, IFN-NR), by [3H]-thymidine-uptake, enzyme-linked immunosorbent assay (ELISA) and Elispot assay and were compared to peptide HBc18 27-specific CTL precursor frequencies among CD8+ T cells derived from HLA-A2+ patients. RESULTS: HBc-specific proliferative PBMC responses and Th frequencies were significantly increased in AHB patients compared with untreated CHB patients. PBMC derived from IFN-R showed stronger cellular responses than IFN-NR. Stimulated PBMC from all patient groups secreted significantly more IFN-gamma than IL-4 indicating Th1/Th0 cell responses. Furthermore, in AHB and IFN-R patients, high peptide HBc18-27-specific CTL precursor frequencies closely correlated with strong HBc-specific Th responses, whereas in untreated CHB and IFN-NR patients lower CTL frequencies were observed without correlation to Th activities. CONCLUSIONS: HBV core-specific Th-cell responses appeared to support efficient CTL induction in patients with viral clearance, whereas in chronic HBV carriers quantitatively insufficient Th and CTL responses were observed. This observation could be important for future therapeutic strategies.

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.

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.

Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.

The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.

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.

Recognition of human renal cell carcinoma and melanoma by HLA-A2-restricted cytotoxic T lymphocytes is mediated by shared peptide epitopes and up-regulated by interferon-gamma.

Cytotoxic T lymphocytes (CTL) have previously been isolated from peripheral blood of patients with renal cell carcinoma (RCC). The CD8-positive CTL line MZ1257-CTL-5 (CTL-5) has been shown to lyse autologous cultured RCC cells in an HLA-A2 restricted fashion. Allogeneic, HLA-A2-matched RCC and melanoma cell lines were also lysed by CTL-5, suggesting that melanoma and renal cancer share antigenic determinants. The aim of the study was to determine whether RCC and melanoma share peptide epitopes that are recognized by CTL-5 in the context of HLA-A2 molecules. Peptides were acideulated from various cell lines, separated by reversed phase high performance liquid chromatography (RP-HPLC), and assessed for their ability to reconstitute the CTL-5-defined epitope by pulsing the peptides on HLA-A2 positive antigen-processing mutant cell line CEM x 721.174.T2 (T2). Peptides eluted from allogeneic HLA-A2-matched RCC and melanoma cell lines exhibited the CTL-5-defined epitope in the same HPLC fractions as peptides derived from the autologous RCC line. Renal cancer and melanoma cells preincubated with interferon-gamma (IFN-gamma) resulted in an additional peak of reconstitution activity in both cell types. This second lytic peak was also observed when high amounts of autologous RCC cells were used for peptide preparation without IFN-gamma pretreatment, indicating that IFN-gamma increases the amount of MHC class I/peptide complexes per cell, rather than inducing a neo-epitope.

Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha 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.

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.