Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T-cells in chronic lymphocytic leukemia.

Antitumor immunity in chronic lymphocytic leukemia (CLL) is hampered by highly dysfunctional T-cells. Although certain T-cell subsets have been reported to be of prognostic significance in this disease, their interplay is complex and it remains incompletely understood which of these subsets significantly drive CLL progression. Here, we determined immunological profiles of 24 circulating T-cell subsets from 79 untreated individuals by multiparametric flow cytometry. This screening cohort included healthy donors, patients with monoclonal B-cell lymphocytosis (MBL), Rai 0 CLL and advanced CLL. We applied multidimensional scaling analysis as rigorous and unbiased statistical tool to globally assess the composition of the circulating T-cell environment and to generate T-cell scores reflecting its integrity. These scores allowed clear distinction between advanced CLL and healthy controls, whereas both MBL and Rai 0 CLL showed intermediate scores mirroring the biological continuum of CLL and its precursor stages. T-cell stimulation and suppression assays as well as longitudinal T-cell profiling showed an increasingly suppressive regulatory function initiating at the MBL stage. Effector function was impaired only after transition to CLL and partially recovered after chemoimmunotherapy. In an independent validation cohort of 52 untreated CLL cases, aberrant T-cell profiles were significantly associated with shorter time to treatment independently of other prognostic parameters. Random forest modeling predicted regulatory T-cell, gamma/delta and NKT-cells, as well as exhaustion of the CD8+ subset as potential drivers of progression. Our data illustrate a pathological T-cell environment in MBL that evolves toward a more and more suppressive and prognostically relevant profile across the disease stages.

NY-CO-58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer.

NY-CO-58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY-CO-58/KIF2C by RT-PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full-length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY-CO-58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki-67 expression (r = 0.69; p = 0.0003). Investigating NY-CO-58/KIF2C-specific T cell responses, CD8(+) T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4(+) T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN-gamma and TNF-alpha. Depletion of CD4(+)CD25(+) T cells before stimulation significantly increased the intensity of the preexisting response. NY-CO-58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY-CO-58/KIF2C was able to induce spontaneous CD4(+) T cell responses of the Th1-type, which were tightly controlled by peripheral T regulatory cells.

Longitudinal analysis and prognostic effect of cancer-testis antigen expression in multiple myeloma.

PURPOSE: Reliable data on the persistence of tumor expression of cancer-testis (CT) antigens over time and consequent analyses of the effect of CT antigen expression on the clinical course of malignancies are crucial for their evaluation as diagnostic markers and immunotherapeutic targets. EXPERIMENTAL DESIGN: Applying conventional reverse transcription-PCR, real-time PCR, and Western blot, we did the first longitudinal study of CT antigen expression in multiple myeloma analyzing 330 bone marrow samples from 129 patients for the expression of four CT antigens (MAGE-C1/CT7, MAGE-C2/CT10, MAGE-A3, and SSX-2). RESULTS: CT antigens were frequently and surprisingly persistently expressed, indicating that down-regulation of these immunogenic targets does not represent a common tumor escape mechanism in myeloma. We observed strong correlations of CT antigen expression levels with the clinical course of myeloma patients as indicated by the number of bone marrow-residing plasma cells and peripheral paraprotein levels, suggesting a role for CT antigens as independent tumor markers. Investigating the prognostic value of CT antigen expression in myeloma patients after allogeneic stem cell transplantation, we found that expression of genes, such as MAGE-C1, represents an important indicator of early relapse and dramatically reduced survival. CONCLUSIONS: Our findings suggest that CT antigens might promote the progression of multiple myeloma and especially MAGE-C1/CT7, which seems to play the role of a "gatekeeper" gene for other CT antigens, might characterize a more malignant phenotype. Importantly, our study also strongly supports the usefulness of CT antigens as diagnostic and prognostic markers as well as therapeutic targets in myeloma.

Signalling via the hypoxia-inducible factor-1alpha requires multiple posttranslational modifications.

Cellular hypoxia, a local decrease in the oxygen concentration below normal (21%) atmospheric concentrations, occurs in both physiological and pathological situations. The transcriptional complex Hypoxia-Inducible Factor-1 (HIF-1) is the key player in the signalling pathway that controls the hypoxic response of mammalian cells. Tight regulation of this response involves posttranslational modification of the alpha subunit of HIF-1. Hydroxylation, ubiquitination, acetylation, S-nitrosation and phosphorylation have been shown to determine its half-life and/or transcriptional activity. The precise spatio-temporal occurrence of these multiple modifications is still not fully understood but is dependent on the microenvironment and determines the driving force of variable cellular responses.

When hypoxia signalling meets the ubiquitin-proteasomal pathway, new targets for cancer therapy.

The ubiquitin-proteasomal pathway of degradation of proteins is activated or repressed in response to a number of environmental stresses and thereby plays an essential role in cell function and survival. Hypoxic stress, resulting from a decrease in the concentration of oxygen in tissues, is encountered in both physiological and pathological situations, in particular in cancer. The transcriptional complex hypoxia-inducible factor (HIF) is the key player in the signalling pathway that controls the hypoxic response of mammalian cells. Under hypoxic conditions it transactivates an impressive number of genes involved in a multitude of cellular functions. Tight regulation of this response in part involves the ubiquitin-proteasomal system where oxygen-dependent prolyl-4-hydroxylation of the alpha subunit of HIF triggers a cascade of events that leads to its degradation by the 26S proteasome. Inhibition of the proteasome in conjunction with topoisomerase inhibition has shown some promise in the treatment of experimental cancer. Such treatment may impact on the hypoxic adaptation of tumour cells.

Multiparameter flow cytometry in the differential diagnosis of aberrant T-cell clones of unclear significance.

Immunophenotypic distinction between neoplastic and reactive T-cell clones can be challenging, as peripheral T-cell lymphomas (PTCLs) lack an immunophenotypic marker of clonality. Systematic screening of 10,510 cases analyzed by immunophenotyping at our institution between 2006 and 2012 resulted in 49 cases with aberrant T-cell populations of unclear significance. Review of patient charts allowed us to assign these cases to three categories. In 21 cases, PTCL could later be confirmed by complementary diagnostics (PTCL group). In 20 cases, follow-up confirmed the reactive nature of the aberrant T-cells (non-PTCL group). Eight cases remained of unclear significance. Neither the population size nor the number of aberrant markers differed significantly between the PTCL and non-PTCL groups. Only loss of CD7 was found significantly more often in patients with PTCL than in patients with non-PTCL (p = 0.037). Our data show that aberrant T-cell populations need to be interpreted in the clinicopathological context, as reactive and neoplastic phenotypes largely overlap.

Decrease of CD4(+)FOXP3(+) T regulatory cells in the peripheral blood of human subjects undergoing a mental stressor.

We have previously shown that acute psychological stress alerts the adaptive immune response causing an increase in antigen-experienced effector T cells in the peripheral blood. T regulatory cells (Tregs) play a central role in maintaining self-tolerance and controlling autoimmune responses. Here, we analyzed for the first time the behaviour of Tregs in the context of a stress-induced activation of the adaptive immune response. 31 healthy young males underwent a brief laboratory stressor and, in a crossover design, served as their own unstressed controls. We quantified effects of acute stress on CD4(+)FOXP3(+) T regulatory cells and other T cell subpopulations using flow cytometry. In addition, the expression of Treg-related effector molecules and stress hormone receptors were analyzed in the subjects' peripheral T cells. We confirmed our previous observation of a stress-induced decrease in CD45RA(+)CCR7(+) "naïve" and CD45RA(-)CCR7+ "central memory" T cells while CD45RA(-)-CCR7(-) "memory effector" and CD45RA(+)CCR7(-) "terminally differentiated" effector T cells remained stable or increased. Importantly, we found acute psychological stress to cause a concomitant decrease in CD4(+)FOXP3(+) Tregs and in CD4(+) T cells expressing Treg-related effector molecules cytotoxic T-lymphocyte antigen-4 (CTLA-4) and latency associated peptide (LAP). Finally, we observed beta(1)-adrenergic and glucorticoid alpha receptors to be overexpressed in Tregs, suggesting that these molecules might mediate stress-related effects on Tregs. In conclusion, inhibiting components of the adaptive immune response, like Tregs, are down-regulated during a stress-induced activation of the adaptive immune response. In situations of chronic stress, this scenario might result in an exacerbation of inflammatory conditions such as autoimmune diseases.

The role of the hypoxia-inducible factor in tumor metabolism growth and invasion.

Oxygen deprivation leading to hypoxia is a common feature of solid tumours. Under these conditions a signalling pathway involving a key oxygen-response regulator termed the hypoxia-inducible factor (HIF) is switched on. HIF is a transcription factor that, in hypoxia, drives the induction or repression of a myriad of genes controlling multiple cell functions such as angiogenesis, metabolism, invasion/metastasis and apoptosis/survival. Thus, the level of oxygen in a cell dictates the molecular response of cells through modulation of gene expression. Here we review the central role of HIF in cancer progression through the tumour response to hypoxia. Within this context the following aspects will be discussed: i) the mechanism by which oxygen deprivation inhibits two oxygen-sensor hydroxylases, thereby releasing the alpha subunit of HIF from programmed destruction by the ubiquitin-proteasome system and from a lock on its transcriptional activity; ii) the way in which the bi-transcriptional activity of HIF-alpha, which is regulated by the interplay between an oxygen-sensor attenuator and co-activators, determines the repertoire of gene expression; and iii) the role that HIF plays in tumour metabolism, in particular in glycolysis, and consequent acidification of the microenvironment, which influences both cell survival and cell death. Finally, the direct link of HIF to tumourigenesis and metastasis will be investigated and approaches for fighting tumour progression through a better understanding of HIF-mediated modulation of tumour metabolism and cell death will be considered.

[Regulation of the Hypoxia-Inducible Factor-1alpha (HIF-1alpha): a breath of fresh air in hypoxia research]. / La regulation de HIF-1alpha (Hypoxia-Inducible Factor-1alpha): un air nouveau dans le domaine de l'hypoxie.

Angiogenesis, a process that leads to the formation of new blood vessels, from a existing network of vessels is tightly regulated. The understanding of mechanisms that control its activity should lead to progress in the treatment of diseases such as cancer and ischemic disorders. In the case of cancer, the rapid growth of tumor cells results in a decrease in the concentration of oxygen, or hypoxia, in the center of the tumor. This stress is the signal that induces angiogenesis. Blood vessels bring nutrients and oxygen to the tumor, allowing it to grow and to metastase. The Hypoxia-Inducible Factor 1, HIF-1, plays a crucial role in this process. HIF-1 is a heterodimer composed of two subunits, alpha and beta. Under hypoxic conditions, HIF-1alpha is stabilized and enters the nucleus, to form a dimer with HIF-1beta, where it induces the expression of its target genes. Among these genes is vegf (vascular endothelial growth factor), a key player in blood vessel formation. The protein HIF-1alpha is subjected to post-translational modifications that are the molecular basis of the hypoxic response although the mechanisms are not completely understood. In this review, we will discuss in particular the multiple post-translational modifications regulating HIF-1alpha activity.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.