BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Cell viability and noninvasive in vivo MRI tracking of 3D cell encapsulating self-assembled microcontainers.

Several molecular therapies require the implantation of cells that secrete biotherapeutic molecules and imaging the location and microenvironment of the cellular implant to ascertain its function. We demonstrate noninvasive in vivo magnetic resonance imaging (MRI) of self-assembled microcontainers that are capable of cell encapsulation. Negative contrast was obtained to discern the microcontainer with MRI; positive contrast was obtained in the complete absence of background signal. MRI on a clinical scanner highlights the translational nature of this research. The microcontainers were loaded with cells that were dispersed in an extracellular matrix, and implanted both subcutaneously and in human tumor xenografts in SCID mice. MRI was performed on the implants, and microcontainers retrieved postimplantation showed cell viability both within and proximal to the implant. The microcontainers are characterized by their small size, three dimensionality, controlled porosity, ease of parallel fabrication, chemical and mechanical stability, and noninvasive traceability in vivo.

Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR.

Efficient immune attack of malignant disease requires the concerted action of both CD8+ CTL and CD4+ Th cells. We used human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic mice, in which the mouse CD8 molecule cannot efficiently interact with the alpha3 domain of A2.1, to generate a high-affinity, CD8-independent T cell receptor (TCR) specific for a commonly expressed, tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the human p53 tumor suppressor protein. Retroviral expression of this CD8-independent, p53-specific TCR into human T cells imparted the CD8+ T lymphocytes with broad tumor-specific CTL activity and turned CD4+ T cells into potent tumor-reactive, p53A2.1-specific Th cells. Both T cell subsets were cooperative and interacted synergistically with dendritic cell intermediates and tumor targets. The intentional redirection of both CD4+ Th cells and CD8+ CTL by the same high-affinity, CD8-independent, tumor-specific TCR could provide the basis for novel broad-spectrum cancer immunotherapeutics.

Recombinant human tumor necrosis factor: an efficient agent for cancer treatment.

Recombinant human TNF (rhTNF) has a selective effect on endothelial cells in tumour angiogenic vessels. Its clinical use has been limited because of its property to induce vascular collapsus. TNF administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs was shown to be safe and efficient. When combined to the alkylating agent melphalan, a single ILP produces a very high objective response rate. ILP with TNF and melphalan provided the proof of concept that a vasculotoxic strategy combined to chemotherapy may produce a strong anti-tumour effect. The registered indication of TNF-based ILP is a regional therapy for regionally spread tumours. In soft tissue sarcomas, it is a limb sparing neoadjuvant treatment and, in melanoma in-transit metastases, a curative treatment. Despite its demonstrated regional efficiency TNF-based ILP is unlikely to have any impact on survival. High TNF dosages induce endothelial cells apoptosis, leading to vascular destruction. However, lower TNF dosage produces a very strong effect that is to increase the drug penetration into the tumour, presumably by decreasing the intratumoural hypertension resulting in better tumour uptake. TNF-ILP allowed the identification of the role of alphaVbeta3 integrin deactivation as an important mechanism of antiangiogenesis. Several recent studies have shown that TNF targeting is possible, paving the way to a new opportunity to administer TNF systemically for improving cancer drug penetration. TNF was the first agent registered for the treatment of cancer that improves drug penetration in tumours and selectively destroys angiogenic vessels.

Chemical modifications of a natural xanthone and antimicrobial activity against multidrug resistant Staphylococcus aureus and cytotoxicity against human tumor cell lines

A series of 15 ω-aminoalkoxylxanthones containing methyl, ethyl, propyl, tert-butylamino and piperidinyl moieties were synthesized from a natural xanthone isolated from a lichen species. These compounds were tested for their in vitro antibacterial properties against Gram-positive and Gram-negative bacteria and cytotoxicity against a number of human tumor cell lines was too evaluated. The newly synthesized derivatives revealed selective activity against Staphylococcus aureus (Gram-positive), and the most promising results are for a multidrug resistant strain, for which six of these compounds showed good activity (MICs 4 µg/mL). Many derivatives inhibited tumor cells growth and most compounds were active on multiple lines.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

N-4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: Cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

N-4-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene) hydrazinecarbothioamide) and its N-4-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N-4-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh), N-4-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N-4-ortho-, -meta- and -para-nitrophenyl (H2Ac4oNO(2)Ph, H2Ac4mNO(2)Ph, H2Ac4pNO(2)Ph) derivatives were assayed for their cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds were highly cytotoxic against the three cell lineages (IC50: MCF-7, 52-0.16 nM; T98G, 140-1.0 nM; U87, 160-1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any haemolytic activity at up to 10(-5) M. The compounds were able to induce programmed cell death. H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization. (C) 2012 Elsevier Ltd. All rights reserved.

Tolerance and immunity to human tumor-associated antigens

Tumor-assoziierte Antigene (TAA) repräsentieren wichtige Zielstrukturen in zytotoxischen T-Zell (ZTL)-basierten Immuntherapien zur Behandlung maligner Erkrankungen. Die Tatsache, dass TAA nicht spezifisch nur in Tumoren sondern auch in nicht-transformierten Zellen vorhanden sind, kann infolge verschiedener Toleranz-Mechanismen zur Eliminierung von ZTL führen, deren T-Zell-Rezeptoren eine hohe Affinität für TAA besitzen. Entsprechend erfordert die Entwicklung effektiver Immuntherapeutika die genaue Analyse des verfügbaren T-Zell-Repertoires mit Spezifität für ein gegebenes TAA.Die Arbeit fokusierte das Tyrosinase (369-377) ZTL-Epitop, das im Komplex mit HLA-A*0201 (A2.1) auf der Zell-Oberfläche von malignen Melanomen und verschiedenen nicht-transformierten Zellen präsentiert wird. Es wurde gefunden, dass sowohl das humane als auch das murine Tyrosinase (369-377)-spezifische ZTL-Repertoire durch Selbst-Toleranz kompromittiert ist und dass diese Toleranz weder durch Verwendung einer bestimmten Peptid-Variante noch durch Interferenz mit CD4+CD25+ regulatorischen T-Zellen oder CTLA-4 umgangen werden kann. Diese Ergebnisse wurden anschließend auf ein anderes Krankheitsmodell, das Multiple Myelom (MM), adaptiert. Unter Umgehung von Selbst-Toleranz in A2.1-transgenen Mäusen wurde gezeigt, dass Transkriptionsfaktoren, die die terminale Differenzierung von B-Zellen in maligne und nicht-maligne Plasmazellen diktieren, als MM-assoziierte ZTL-Epitope dienen können.Diese Arbeit bietet einen bedeutenden und innovativen Beitrag zur Gestaltung von Tyrosinase-basierten Melanom- und MM-reaktiven Immuntherapien.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

Reliability of intraoperative high-resolution 2D ultrasound as an alternative to high-field strength MR imaging for tumor resection control: a prospective comparative study

OBJECT: Ultrasound may be a reliable but simpler alternative to intraoperative MR imaging (iMR imaging) for tumor resection control. However, its reliability in the detection of tumor remnants has not been definitely proven. The aim of the study was to compare high-field iMR imaging (1.5 T) and high-resolution 2D ultrasound in terms of tumor resection control. METHODS: A prospective comparative study of 26 consecutive patients was performed. The following parameters were compared: the existence of tumor remnants after presumed radical removal and the quality of the images. Tumor remnants were categorized as: detectable with both imaging modalities or visible only with 1 modality. RESULTS: Tumor remnants were detected in 21 cases (80.8%) with iMR imaging. All large remnants were demonstrated with both modalities, and their image quality was good. Two-dimensional ultrasound was not as effective in detecting remnants<1 cm. Two remnants detected with iMR imaging were missed by ultrasound. In 2 cases suspicious signals visible only on ultrasound images were misinterpreted as remnants but turned out to be a blood clot and peritumoral parenchyma. The average time for acquisition of an ultrasound image was 2 minutes, whereas that for an iMR image was approximately 10 minutes. Neither modality resulted in any procedure-related complications or morbidity. CONCLUSIONS: Intraoperative MR imaging is more precise in detecting small tumor remnants than 2D ultrasound. Nevertheless, the latter may be used as a less expensive and less time-consuming alternative that provides almost real-time feedback information. Its accuracy is highest in case of more confined, deeply located remnants. In cases of more superficially located remnants, its role is more limited.

COMPARISON OF DNA TRANSFECTION AND MICROCELL-MEDIATED CHROMOSOME TRANSFER FOR DETECTING TRANSFORMING GENES IN HUMAN TUMOR CELL LINES

DNA mediated gene transfection is an important tool for moving and isolating genes from one cell type and putting them into a foreign genetic background. DNA transfection studies have been done routinely in many laboratories to identify and isolate transforming sequences in human tumors and tumor cell lines. A second technique, microcell-mediated chromosome transfer, allows the transfer of small numbers of intact human chromosome from one cell to another. This work was done to compare the efficiency of these two techniques in the transformation of NIH 3T3 mouse fibroblast cells.^ My intent in comparing these two techniques was to see if there was a difference in the transforming capability of DNA which has been purified of all associated protein and RNAs, and that of DNA which is introduced into a cell in its native form, the chromosome. If chromosomal sequences were capable of transforming the 3T3 cells in culture, the method could then be used as a way to isolate the relevant tumorigenic chromosomes from human tumors.^ The study shows, however, that even for those cell lines that contain transforming sequences identified by DNA-mediated gene transfer, those same sequences were unable to transform 3T3 cells when introduced to the cells by somatic fusion of human tumor microcells. I believe that the human transforming sequences in their original genetic conformation are not recognized by the mouse cell as genes which should be expressed; therefore, no noticeable transformation event was selected by this technique. ^

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.