Our results suggest a significant association between recent infection and CAD that is not explained by mechanical factors occurring during infection.

Key Points HLA mismatches at the allele and antigen level (possibly with the exception of HLA-DQB1) should be treated equally in donor selection. HLA mismatches at >1 locus (including HLA-DQB1) have additive detrimental effects.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA=B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA=B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA=B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA=B. Pharmacological inhibition of metalloproteases reduced the level of released MICA=B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and=or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA=B transfection systems.The activating natural killer group 2, member D (NKG2D) receptor is expressed on NK cells, NKT cells, gd T cells, CD8 1 T cells and a minor immunoregulatory subset of CD4 1 T cells. 1,2 The ligation of NKG2D costimulates T cells but also directly triggers cell-mediated cytotoxicity and cytokine release in NK cells. [3][4][5][6] Thus, despite some exceptions including gastrointestinal epithelium 7 and placenta, 8 the ligands for NKG2D are not expressed on healthy tissues to avoid inadvertent damage. However, NKG2D ligand (NKG2DL) expression can be induced in response to a variety of stimuli related to cellular stress such as heat shock, viral infection, DNA damage, oxidative stress and certain proinflammatory signals. 9,10 Of note, many tumors and precancerous lesions express NKG2D ligands presumably as a bystander effect of the oncogenic process itself. 11 As NKG2DL expression renders tumors susceptible to NKG2D 1 lymphocytes, the NKG2D system plays a pivotal role in immunosurveillance. 12 On the other hand, the sustained exposure to tumor cell-bound NKG2D ligands might also result in surface modulation and functional impairment of the NKG2D receptor and induce cross-tolerance of additional NK cell activation pathways. 13,14 A hallmark of the NKG2D system is the multitude of ligands identified so far. In humans, these include the MHC class I-related chain ...

Infection with high-risk human papillomavirus (HPV) is necessary for the development of a cervical lesion, but only a fraction of precursor lesions progress to cancer. Additional factors, other than HPV type per se, are likely to increase the probability for progression. Intratype genome variations have been reported to be associated with viral persistence and the development of a major cervical disease. We have recently shown that the prevalence of specific HPV16-E6 variants in invasive cervical cancer (ICC) varies between Italian and Swedish women. To extend our initial study we have analyzed E6 variants in cervical lesions from Czech women, ranging from low-grade cervical intraepithelial neoplasia (LCIN) to ICC and scaled up the sample size of our initial study of Swedish and Italian women. In addition, we have correlated the cases of cancers with human leukocyte antigen (HLA) class II haplotypes. In line with our earlier observation, the distribution of specific HPV16-E6 genotypes in CIN and ICC varied in the 3 cohorts. For instance, the HPV16-E6 L83V variant, which has been found to be positively associated with ICC in Swedish women (p ‫؍‬ 0.002), was more prevalent in LCIN than in ICC in Italian and Czech women (p ‫؍‬ 0.01 and ‫؍‬ 0.03, respectively). These data indicate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16-E6 L83V variant. Indeed, the DR04-DQ03 haplotype, which is approximately 3-fold more abundant in the normal Swedish population than in those in Italy and the Czech Republic, was found to be positively associated with HPV16-E6 L83V in the 3 cohorts investigated (p ‫؍‬ 0.01). This observation may explain why L83V is a risk factor more in Sweden than in the other 2 countries. © 2001 Wiley-Liss, Inc. Key words: HPV16; E6 and E7 genotypes; polymorphism; cervical lesion; human leukocyte antigenCertain human papillomavirus (HPV) genotypes are etiologic agents for cervical cancer development. 1 HPV16 is the most frequently detected genotype in cervical carcinoma and its precursors. [2][3][4][5] Biochemical data have demonstrated that the products of 2 early genes, E6 and E7, are the major oncoproteins of HPV16. 6 HPV16 E6 and E7 associate with and inactivate the biologic function of several cellular proteins, including the regulators of cell cycle checkpoints, p53 and pRb. 7 Infection with high-risk HPV is the principal risk factor for the development of an invasive lesion. However, most HPV infections regress spontaneously and, for the cases that do progress to cancer, a long period of latency is normally required. It is clear that additional risk factors play a role in disease progression. Recently, independent studies have provided evidence that specific intratype HPV genome variations may influence the persistence of the infection and the progression of a precursor lesion to cancer. 8 -10 Several North American and South American studies have shown that nonprototype-like HPV16 variants, which harbor several nucleotide changes within the...

Mutations in the nucleophosmin gene (NPM1 mut ) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1 mut . In the present study, we were able to demonstrate both CD4 ؉ and CD8 ؉ T-cell responses against NPM1 mut . Ten peptides derived from wild-type NPM1 and NPM1 mut were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients.Tetramer assays against the most interesting epitopes were performed and Cr 51 -release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4 ؉ T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1 mut induced CD8 ؉ T-cell responses. A total of 33% of the NPM1 mut AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4 ؉ T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8 ؉ and CD4 ؉ T cells. The results of the present study show that NPM1 mut induces specific T-cell responses of CD4 ؉ and CD8 ؉ T cells and therefore is a promising target for specific immunotherapies in AML. (Blood. 2012;120(6):1282-1289) IntroductionMutations in the nucleophosmin 1 gene (NPM1 mut ) represent some of the most common gene mutations in acute myeloid leukemia (AML). 1,2 Falini et al first described the abnormal cytoplasmic localization of the NPM1 protein caused by mutations in exon 12 of the gene. 3 In AML patients with normal cytogenetics, the incidence of NPM1 mut was reported in up to 60% of the patients. 1-3 NPM1 constitutes an important prognostic marker, especially in the context of FMS-related tyrosine kinase internal tandem duplication (FLT3-ITD). In more than 90% of AML patients harboring NPM1 mut , the 3 different NPM1 mut types (A, B, and D) were found. 1,2,4-6 AML patients harboring an NPM1 mut without an FLT3-ITD mutation showed improved survival when treated with intensive chemotherapy. 2 Most AML patients with NPM1 mut seem not to benefit from an allogeneic stem cell transplantation as a first-line treatment. 2,7 However, this issue remains to be elucidated in the context of minimal residual disease detection 8 and the coexistence of other molecular markers. The functional role of NPM1 mut for the improved clinical outcome remains to be elucidated. Immune responses may contribute to clinical outcome by lysis of residual leukemic cells through specific T cells after chemotherapy. Leukemia-associated antigens (LAAs) can be targeted by the immune system in a specific manner, leading to the hypothesis that the expression of LAAs might also influence the clinical outcome of AML patients. mRNA expression of at least 1 of the 3 LAA, receptor for hyaluronic acid-mediated motility (RHAMM), preferentially expressed antigen in mela...

Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor recognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I-related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM) cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is upregulated upon chemotherapeutic treatment both in patient-derived CD138(+)/CD38(+) plasma cells and in several MM cell lines, and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs and metalloproteinase inhibitors enhances NK cell-mediated recognition of MM cells, preserving MIC molecules on the cell surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell-based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune responses

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Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.