Analysis of viral- and tumor antigen-specific CD4 T cell responses in healthy donors and melanoma patients

Résumé Des tentatives pour développer des traitements anti-cancéreux basés sur l'utilisation d'antigènes tumoraux ont commencé il y a plus de 10 ans. Depuis quelques années, un certain intérêt s'est portée sur une sous-population particulière des cellules du système immunitaire, les lymphocytes T CD4. Ces cellules jouent un rôle central dans les réponses immunitaires tant contre les virus que contre les cellules tumorales. Comme d'autres lymphocytes T, ces cellules sont activées de manière spécifique en reconnaissant un morceau d'antigène, appelé peptide. Ces peptides proviennent soit de protéines des cellules de l'hôte, soit des protéines étrangères (virus ou bactéries) soit de cellules transformées (cellules tumorales) et sont présentés aux lymphocytes T par des molécules du soi appelées CMH (complexe majeur d'histocompatibilité). Dans le cas des lymphocytes T CD4, ces molécules sont plus précisément des molécules du CMH de classe II (CMH II). Mis à part l'intérêt porté aux réponses médiées par les lymphocytes T cytotoxiques, un intérêt croissant pour les lymphocytes T CD4 s'est développé à cause de la place centrale qu'occupent ces cellules dans les réponses immunitaires. L'identification d'épitopes présentés par des molécules du CMH de classe II dérivés d'un grand nombre d'antigènes tumoraux, ainsi que le développement de techniques permettant de suivre les réponses immunitaires, offre des opportunités pour étudier de manière quantitative et qualitative les lymphocytes T CD4 spécifiques pour un antigène particulier chez des patients cancéreux. De plus, ces épitopes permettent d'induire des réponses médiées par les lymphocytes T CD4 et CD8 chez ces mêmes patients. Dans ce travail, notre premier but était de valider l'utilisation de multimères formés par des complexes peptide:molécules de CMH de class II (pCMH II) pour quantifier la réponse des cellules T CD4 dirigée contre l'épitope HA307-319 dérivé de la protéine hémaglutinine du virus de la grippe et présenté par HLA-DRB1*0401. En analysant des échantillons provenant de volontaires sains ayant reçus un vaccin contre la grippe, nous avons pu démontrer une expansion et une activation transitoires des lymphocytes T CD4 spécifiques pour le peptide HA307-319 après vaccination. De plus, les multimères pCMH II nous ont permis d'analyser plus en détails hétérogénéité des cellules T CD4 spécifiques pour le peptide HA307-319 présents dans le sang périphérique d'individus sains. Par la suite, notre but a été d'analyser les réponses des lymphocytes T CD4 spécifiques pour l'antigène Melan-A chez des patients atteints de mélanome métastatique. Nous avons tout d'abord démontré la présence de cellules T CD4 spécifiques pour l'épitope Melan-A51-73, présenté par HLA-DRBl*0401, qui avait déjà été préalablement décrit. Ensuite, nous avons décrit et caractérisé 2 nouveaux peptides issus de Melan-A qui sont présentés aux cellules T CD4 par différentes molécules du CMH de clans II. Des cellules spécifiques pour ces deux épitopes ont été trouvées chez 9/ 16 patients analysés. De plus, des multimères pCMH II chargés avec un des épitopes nous ont permis de détecter ex vivo des lymphocytes T CD4 spécifiques pour Melan-A dans le sang périphérique d'un patient atteint de mélanome. Mis ensemble, tous ces résultats suggèrent une potentielle utilisation des multimères pCMH II pour analyser en détail les lymphocytes T CD4 spécifiques d'antigènes définis. Cependant, le suivi ex vivo de telles cellules ne semble être possible que dans des cas bien particuliers. Néanmoins, les nouveaux épitopes issus de Melan-A et présentés par des molécules du CMH de classe II que nous avons décrits dans cette étude aideront à étudier plus en détails les lymphocytes T CD4 spécifiques pour Melan-A chez des patients atteints de mélanome, un sujet d'étude sur lequel peu de résultats sont à ce jour disponibles. Summary Attempts to develop cancer vaccines based on molecularly defined tumorassociated antigens were initiated more than 10 years ago. Apart from CTLmediated anti-tumor immunity, interests are. now focused on CD4 T cells that are central players of immune responses. The identification of MHC class-II-restricted epitopes from numerous tumor antigens together with the development of monitoring tools offers the opportunity to quantitatively and qualitatively study antigen-specific CD4 T lymphocytes in cancer patients and to induce both CTL and T helper responses in cancer patients. In this work, we first aimed at validating the use of peptide:MHC class II complex (pMHC II) multimers to quantitate the CD4 T cell response against the hemagglutinin-derived epitope HAso~-si9 from influenza virus presented by HLA-DRBl*0401. By analysing samples from healthy volunteers vaccinated with ananti-influenza vaccine, we could demonstrate a transient expansion and activation of HA-specific CD4 T cells after treatment. Moreover, pMHC II multimers helped us to study the heterogeneity of HAspecific CD4 T cells found in peripheral blood of healthy individuals. Then, we aimed to analyse Melan-A-specific CD4 T cell responses in metastatic melanoma patients. We first demonstrated the presence of CD4 T cells specific for the previously described Melan-A51_73 epitope presented by HLA-DRB 1 *0401 in peripheral blood of those patients. Second, we described and characterised 2 new Melan-A-derived peptides that are presented by different MHC II molecules to CD4 T cells. Specific cells for these epitopes were found in 9/ 16 rnelánoma patients analysed. In addition, pMHC II multimers loaded with one of the two epitopes allowed us to detect ex vivo Melan-A-specific CD4 T cells in peripheral blood of a melanoma patient. Together, these results suggest a potential use of pMHC II multimers in analysing in detail antigen-specific CD4 T cells. However, ex vivo monitoring of such cells will be possible only in particular conditions. Nevertheless, the new Melan-A-derived MHC II-restricted epitopes described here will help to study in more detail Melan-A-specific CD4 T cells in melanoma patients, a field where only scarce data are available.

Expression of common acute lymphoblastic leukemia antigen (CALLA) on human malignant melanoma cell lines.

Fifteen human melanoma cells lines were tested by an antibody-binding radioimmunoassay using a monoclonal antibody (A12) directed against the common acute lymphoblastic leukemia antigen (CALLA). Cells from six melanoma lines were found to react with this antibody. The level of antigen and the percentage of positive cells in these six melanoma lines showed wide variation, as demonstrated by analysis in the fluorescence-activated cell sorter (FACS). Immunoprecipitation of solubilized 125I-labeled membrane proteins from CALLA positive melanoma cells with A12 monoclonal antibody revealed a major polypeptide chain with an apparent m.w. of 100,000 daltons, characteristic for CALLA as determined on SDS-polyacrylamide gel electrophoresis. The expression of CALLA on MP-6 melanoma cells was modulated when the cells were cultured in the presence of A12 antibody. Reexpression of CALLA on these cells occurred within 5 days after transfer of the modulated cells into medium devoid of monoclonal antibody.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Population-based registration of phenotypic anatomic and familial data for melanoma: results from a Swiss multicentric study

CONTEXT AND OBJECTIVES: A multicentric study was set up to assess the feasibility for Swiss cancer registries of actively retrieving 3 additional variables of epidemiological and a etiological relevance for melanoma, and of potential use for the evaluation of prevention campaigns. MATERIAL AND METHODS: The skin type, family history of melanoma and precise anatomical site were retrieved for melanoma cases registered in 5 Swiss cantons (Neuchâtel, St-Gall and Appenzell, Vaud and Wallis) over 3 to 6 consecutive years (1995-2002). Data were obtained via a short questionnaire administered by the physicians - mostly dermatologists - who originally excised the lesions. As the detailed body site was routinely collected in Ticino, data from this Cancer Registry were included in the body site analysis. Relative melanoma density (RMD) was computed by the ratio of observed to expected numbers of melanomas allowing for body site surface areas, and further adjusted for site-specific melanocyte density. RESULTS: Of the 1,645 questionnaires sent, 1,420 (86.3%) were returned. The detailed cutaneous site and skin type were reliably obtained for 84.7% and 78.7% of questionnaires, and family history was known in 76% of instances. Prevalence of sun-sensitive subjects and patients with melanoma affected first-degree relatives, two target groups for early detection and surveillance campaigns were 54.1% and 3.4%, respectively. After translation into the 4th digit of the International Classification of Diseases for Oncology, the anatomical site codes from printed (original information) and pictorial support (body chart from the questionnaire) concurred for 94.6% of lesions. Discrepancies occurred mostly for lesions on the upper, outer part of the shoulder for which the clinician's textual description was "shoulder blade". This differential misclassification suggests under-estimation by about 10% of melanomas of the upper limbs and an over-estimation of 5% for truncal melanomas. Sites of highest melanoma risk were the face, the shoulder and the upper arm for sexes, the back for men and the leg for women. Three major features of this series were: (1) an unexpectedly high RMD for the face in women (6.2 vs 4.2 in men), (2) the absence of a male predominance for melanomas on the ears, and (3) for the upper limbs, a steady gradient of increasing melanoma density with increasing proximity to the trunk, regardless of sex. DISCUSSION AND CONCLUSION: The feasibility of retrieving the skin type, the precise anatomical location and family history of melanoma in a reliable manner was demonstrated thanks to the collaboration of Swiss dermatologists. Use of a schematic body drawing improves the quality of the anatomical site data and facilitate the reporting task of doctors. Age and sex patterns of RMD paralleled general indicators of sun exposure and behaviour, except for the hand (RMD=0.2). These Swiss results support some site or sun exposure specificity in the aetiology of melanoma.

Complete and long-lasting regression of disseminated multiple skin melanoma metastases under treatment with cyclooxygenase-2 inhibitor.

Experimental and clinical evidence indicates that non-steroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors may have anti-cancer activities. Here we report on a patient with a metastatic melanoma of the leg who experienced a complete and sustained regression of skin metastases upon continuous single treatment with the cyclooxygenase-2 inhibitor rofecoxib. Our observations indicate that the inhibition of cyclooxygenase-2 can lead to the regression of disseminated skin melanoma metastases, even after failure of chemotherapy.

Left-sided excess of melanoma occurrence but not of other skin cancers: additional evidence.

Comment on: Butler ST, Fosko SW. Increased prevalence of left-sided skin cancers. J Am Acad Dermatol. 2010 Dec;63(6):1006-10. PMID: 20226568.

Silver-assisted laser desorption ionization for high spatial resolution imaging mass spectrometry of olefins from thin tissue sections.

Silver has been demonstrated to be a powerful cationization agent in mass spectrometry (MS) for various olefinic species such as cholesterol and fatty acids. This work explores the utility of metallic silver sputtering on tissue sections for high resolution imaging mass spectrometry (IMS) of olefins by laser desorption ionization (LDI). For this purpose, sputtered silver coating thickness was optimized on an assorted selection of mouse and rat tissues including brain, kidney, liver, and testis. For mouse brain tissue section, the thickness was adjusted to 23 ± 2 nm of silver to prevent ion suppression effects associated with a higher cholesterol and lipid content. On all other tissues, a thickness of at 16 ± 2 nm provided the best desorption/ionization efficiency. Characterization of the species by MS/MS showed a wide variety of olefinic compounds allowing the IMS of different lipid classes including cholesterol, arachidonic acid, docosahexaenoic acid, and triacylglyceride 52:3. A range of spatial resolutions for IMS were investigated from 150 μm down to the high resolution cellular range at 5 μm. The applicability of direct on-tissue silver sputtering to LDI-IMS of cholesterol and other olefinic compounds presents a novel approach to improve the amount of information that can be obtained from tissue sections. This IMS strategy is thus of interest for providing new biological insights on the role of cholesterol and other olefins in physiological pathways or disease.

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.

The paradoxical coexistence of spontaneous tumor antigen-specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1-specific CD8(+) T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1-specific CD8(+) T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1(+) NY-ESO-1-specific CD8(+) T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3(+)PD-1(+) NY-ESO-1-specific CD8(+) T cells are more dysfunctional than Tim-3(-)PD-1(+) and Tim-3(-)PD-1(-) NY-ESO-1-specific CD8(+) T cells, producing less IFN-γ, TNF, and IL-2. Tim-3-Tim-3L blockade enhanced cytokine production by NY-ESO-1-specific CD8(+) T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3-Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1-specific CD8(+) T cells upon prolonged antigen stimulation and acted in synergy with PD-1-PD-L1 blockade. Collectively, our findings support the use of Tim-3-Tim-3L blockade together with PD-1-PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

Cloning of an interferon-gamma regulated human melanoma-associated antigen: identity to the intercellular adhesion molecule ICAM-1.

The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.

Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab.

Metastatic melanoma has a poor prognosis with high resistance to chemotherapy and radiation. Recently, the anti-CTLA-4 antibody ipilimumab has demonstrated clinical efficacy, being the first agent to significantly prolong the overall survival of inoperable stage III/IV melanoma patients. A major aim of patient immune monitoring is the identification of biomarkers that predict clinical outcome. We studied circulating myeloid-derived suppressor cells (MDSC) in ipilimumab-treated patients to detect alterations in the myeloid cell compartment and possible correlations with clinical outcome. Lin(-) CD14(+) HLA-DR(-) monocytic MDSC were enriched in peripheral blood of melanoma patients compared to healthy donors (HD). Tumor resection did not significantly alter MDSC frequencies. During ipilimumab treatment, MDSC frequencies did not change significantly compared to baseline levels. We observed high inter-patient differences. MDSC frequencies in ipilimumab-treated patients were independent of baseline serum lactate dehydrogenase levels but tended to increase in patients with severe metastatic disease (M1c) compared to patients with metastases in skin or lymph nodes only (M1a), who had frequencies comparable to HD. Interestingly, clinical responders to ipilimumab therapy showed significantly less lin(-) CD14(+) HLA-DR(-) cells as compared to non-responders. The data suggest that the frequency of monocytic MDSC may be used as predictive marker of response, as low frequencies identify patients more likely benefitting from ipilimumab treatment. Prospective clinical trials assessing MDSC frequencies as potential biomarkers are warranted to validate these observations.

Optical response of Cu3Ge thin films

We report an investigation on the optical properties of Cu3Ge thin films displaying very high conductivity, with thickness ranging from 200 to 2000 Å, deposited on Ge substrates. Reflectance, transmittance, and ellipsometric spectroscopy measurements were performed at room temperature in the 0.01-6.0, 0.01-0.6, and 1.4-5.0 eV energy range, respectively. The complex dielectric function, the optical conductivity, the energy-loss function, and the effective charge density were obtained over the whole spectral range. The low-energy free-carrier response was well fitted by using the classical Drude-Lorentz dielectric function. A simple two-band model allowed the resulting optical parameters to be interpreted coherently with those previously obtained from transport measurements, hence yielding the densities and the effective masses of electrons and holes.

Perpendicular magnetic anisotropy in chemically disordered FePd-FeV(100) alloy thin films

We find that the use of V(100) buffer layers on MgO(001) substrates for the epitaxy of FePd binary alloys yields to the formation at intermediate and high deposition temperatures of a FePd¿FeV mixed phase due to strong V diffusion accompanied by a loss of layer continuity and strong increase of its mosaic spread. Contrary to what is usually found in this kind of systems, these mixed phase structures exhibit perpendicular magnetic anisotropy (PMA) which is not correlated with the presence of chemical order, almost totally absent in all the fabricated structures, even at deposition temperatures where it is usually obtained with other buffer layers. Thus the observed PMA can be ascribed to the V interdiffusion and the formation of a FeV alloy, being the global sample saturation magnetization also reduced.

Nanoscale capacitance microscopy of thin dielectric films

We present an analytical model to interpret nanoscale capacitance microscopy measurements on thin dielectric films. The model displays a logarithmic dependence on the tip-sample distance and on the film thickness-dielectric constant ratio and shows an excellent agreement with finite-element numerical simulations and experimental results on a broad range of values. Based on these results, we discuss the capabilities of nanoscale capacitance microscopy for the quantitative extraction of the dielectric constant and the thickness of thin dielectric films at the nanoscale.

Cationic Diffusion in La2/3Ca1/3MnO3 Thin Films Grown on LaAlO3 (001) Substrates.

Microstructural features of La2/3Ca1/3MnO3 layers of various thicknesses grown on top of 001 LaAlO3 substrates are studied by using transmission electron microscopy and electron energy loss spectroscopy. Films are of high microstructural quality but exhibit some structural relaxation and mosaicity both when increasing thickness or after annealing processes. The existence of a cationic segregation process of La atoms toward free surface has been detected, as well as a Mn oxidation state variation through layer thickness. La diffusion would lead to a Mn valence change and, in turn, to reduced magnetization.