The loss of telomere function can result in telomeric fusion events that lead to the types of genomic rearrangements, such as nonreciprocal translocations, that typify early-stage carcinogenesis. By using single-molecule approaches to characterize fusion events, we provide a functional definition of fusogenic telomeres in human cells. We show that approximately half of the fusion events contained no canonical telomere repeats at the fusion point; of those that did, the longest was 12.8 repeats. Furthermore, in addition to end-replication losses, human telomeres are subjected to large-scale deletion events that occur in the presence or absence of telomerase. Here we show that these telomeres are fusogenic, and thus despite the majority of telomeres being maintained at a stable length in normal human cells, a subset of stochastically shortened telomeres can potentially cause chromosomal instability. Telomere fusion was accompanied by the deletion of one or both telomeres extending several kilobases into the telomere-adjacent DNA, and microhomology was observed at the fusion points. This contrasted with telomere fusion that was observed following the experimental disruption of TRF2. The distinct error-prone mutational profile of fusion between critically shortened telomeres in human cells was reminiscent of Ku-independent microhomology-mediated end-joining.[Keywords: Telomere; telomerase; genomic instability; mutation; DNA repair; neoplasia] Supplemental material is available at http://www.genesdev.org.

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Purpose: Patients with chronic lymphocytic leukemia (CLL) display immune deficiency that is most obvious in advanced stage disease. Here we investigated whether this immune dysfunction plays a pathologic role in the progression of early stage disease patients.Experimental Design: We carried out eight-color immunophenotyping analysis in a cohort of 110 untreated early stage CLL patients and 22 age-matched healthy donors and correlated our findings with clinical outcome data.Results: We found a significant reduction in naive CD4 þ and CD8 þ T cells in CLL patients. Only the CD4 þ subset showed significantly increased effector memory cells (T EM and T EMRA ) in the whole cohort (P ¼ 0.004 and P ¼ 0.04, respectively). However, patients with inverted CD4:CD8 ratios (52 of 110) showed preferential expansion of the CD8 compartment, with a skewing of CD8 þ T EMRA (P ¼ 0.03) coupled with increased percentage of CD57 þ CD28 À CD27 À T cells (P ¼ 0.008) and PD-1 positivity (P ¼ 0.027), consistent with a replicative senescence phenotype. Furthermore, inverted CD4:CD8 ratios were associated with shorter lymphocyte doubling time (P ¼ 0.03), shorter time to first treatment (P ¼ 0.03), and reduced progression-free survival (P ¼ 0.005). Conclusions: Our data show that the emergence of CD8 þ PD-1 þ replicative senescence phenotype in early stage CLL patients is associated with more aggressive clinical disease. Importantly, these findings were independent of tumor cell prognostic markers and could not be accounted for by patient age, changes in regulatory T-cell frequency, or cytomegalovirus serostatus. Clin Cancer Res; 18(3); 678-87. Ó2011 AACR.

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.

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Upregulation of the immunosuppressive cell surface glycoprotein, CD200, is a common feature of acute myeloid leukemia (AML) and is associated with poor patient outcome. We investigated whether CD200 overexpression on AML cells could specifically compromise patient natural killer (NK) cell anti-tumor responses. We found that CD200hi patients showed a 50% reduction in the frequency of activated NK cells (CD56dimCD16+) compared with CD200lo patients. Additionally, NK receptor expression (NKp44 and NKp46) on these cells was also significantly downregulated in CD200hi patients. To assess whether NK cell activity was directly influenced by CD200 expression, we examined the effect of ectopic expression of CD200. These assays revealed that both NK cell cytolytic activity and interferon-γ response were significantly reduced toward CD200+ leukemic targets and that these targets showed increased survival compared with CD200− cells. Similarly, NK cells isolated from AML patients were less functionally active toward CD200hi autologous blasts from both cytolytic and immunoregulatory perspectives. Finally, blocking CD200 alone was sufficient to recover a significant proportion of NK cell cytolytic activity. Together, these findings provide the first evidence that CD200 has a direct and significant suppressive influence on NK cell activity in AML patients and may contribute to the increased relapse rate in CD200+ patients.

Summary:Quantitative competitive PCR was used to monitor the quantity of cytomegalovirus (HCMV) in 1647 blood samples from 110 BMT recipients. DNAemia was detected in 49/110 (45%) of the patients, of whom 15/49 experienced HCMV disease. Peak virus load during surveillance was elevated in symptomatic (median 4.5 log 10 genomes/ml) vs asymptomatic patients (median 3.6 log 10 genomes/ml, P = 0.002) and was also significantly elevated in HCMV seropositive recipients of seronegative marrow, (R+D−, median 5.0 log 10 ), compared to those in the R−D− and R+D+ groups (P Ͻ 0.01 and Ͻ0.005). Odds ratios for disease per 0.25 log 10 increase in viral load, recipient seropositivity and aGVHD were 1.43 (P = 0.004), 6.60 (P = 0.05) and 3.17 (P = 0.08), respectively. In multivariate logistic regression analysis only elevated viral load remained a significant risk factor for HCMV disease. The computed disease probability viral load curve showed a rapid increase in disease risk at viral loads between 3.8 and 5.5 log 10 genomes/ml in blood, and odds ratios for disease were determined for different threshold viral loads. These data demonstrate the central role of viral load in the pathogenesis of HCMV in BMT recipients and provide an additional marker for targeting and monitoring therapy.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E629–38 that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201+ HPV16 E6+ cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E629–38 correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-γ, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E629–38 epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E629–38 epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.