Identification of human leukocyte antigen (HLA) types from DNA-sequenced human samples is important in organ transplantation and cancer immunotherapy and remains a challenging task considering ...sequence homology and extreme polymorphism of HLA genes. We present Orthanq, a novel statistical model and corresponding application for transparent and uncertainty-aware quantification of haplotypes. We utilize our approach to perform HLA typing while, for the first time, reporting uncertainty of predictions and transparently observing mutations beyond reported HLA types. Using 99 gold standard samples from 1000 Genomes, Illumina Platinum Genomes and Genome In a Bottle projects, we show that Orthanq can provide overall superior accuracy and shorter runtimes than state-of-the-art HLA typers. Orthanq is the first approach that allows to directly utilize existing pangenome alignments and type all HLA loci. Moreover, it can be generalized for usages beyond HLA typing, e.g. for virus lineage quantification. Orthanq is available under https://orthanq.github.io.
Natural killer (NK) cells are potent immune effector cells capable of mediating antitumor responses. Thus, during immunoediting, tumor cell populations evolve strategies to escape NK-cell-mediated ...recognition. In this study, we report a novel mechanism of immune escape involving tumor cell shedding of B7-H6, a ligand for the activating receptor NKp30 that mediates NK-cell binding and NK-cell-mediated killing. Tumor cells from different cancer entities released B7-H6 by ectodomain shedding mediated by the cell surface proteases "a disintegrin and metalloproteases" (ADAM)-10 and ADAM-17, as demonstrated through the use of pharmacologic inhibitors or siRNA-mediated gene attenuation. Inhibiting this proteolytic shedding process increased the levels of B7-H6 expressed on the surface of tumor cells, enhancing NKp30-mediated activation of NK cells. Notably, we documented elevated levels of soluble B7-H6 levels in blood sera obtained from a subset of patients with malignant melanoma, compared with healthy control individuals, along with evidence of elevated B7-H6 expression in melanoma specimens in situ. Taken together, our results illustrated a novel mechanism of immune escape in which tumor cells impede NK-mediated recognition by metalloprotease-mediated shedding of B7-H6. One implication of our findings is that therapeutic inhibition of specific metalloproteases may help support NK-cell-based cancer therapy.
Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8
T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on ...tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.
Abstract
Poor clinical responses to checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies in melanoma have recently been associated with acquired IFNγ resistance that protects tumor cells ...from the antiproliferative and pro-apoptotic cytokine activity. IFNγ-resistant melanoma cells very often lack functional expression of the IFNγ signaling pathway gene JAK2 due to gene deletions or inactivating gene mutations. Analyzing melanoma cell lines (n = 46, applying next-generation targeted sequencing and single nucleotide polymorphism arrays) as well as available genomic data sets from The Cancer Genome Atlas (TCGA) tumor tissue samples (cutaneous melanoma n = 367, lung squamous cell carcinoma n = 501, bladder urothelial carcinoma n = 408, breast invasive carcinoma n = 768, colorectal adenocarcinoma n = 257), we demonstrate that the frequent chromosomal losses of the tumor suppressor CDKN2A in melanoma and other tumor entities enhance the susceptibility to IFNγ resistance by concomitant deletion of the JAK2 gene (odds ratio = 223.17, 95% confidence interval = 66.91 to 1487.38, two-sided P = 7.6×10-46). Tumors with JAK2 mutations or homozygous JAK2 deletions demonstrate allelic losses covering both CDKN2A and JAK2. This suggests that patients with tumor chromosomal CDKN2A losses are susceptible to developing immunotherapy resistance and should be screened for JAK2 deficiency prior to and under immune checkpoint blocking therapy.
Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the ...host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model. In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins. In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.
CD4
T cells play an important role in immune responses against pathogens and cancer cells. Although their main task is to provide help to other effector immune cells, a growing number of infections ...and cancer entities have been described in which CD4
T cells exhibit direct effector functions against infected or transformed cells. The most important cell type in this context are cytotoxic CD4
T cells (CD4
CTL). In infectious diseases anti-viral CD4
CTL are mainly found in chronic viral infections. Here, they often compensate for incomplete or exhausted CD8
CTL responses. The induction of CD4
CTL is counter-regulated by Tregs, most likely because they can be dangerous inducers of immunopathology. In viral infections, CD4
CTL often kill via the Fas/FasL pathway, but they can also facilitate the exocytosis pathway of killing. Thus, they are very important effectors to keep persistent virus in check and guarantee host survival. In contrast to viral infections CD4
CTL attracted attention as direct anti-tumor effectors in solid cancers only recently. Anti-tumor CD4
CTL are defined by the expression of cytolytic markers and have been detected within the lymphocyte infiltrates of different human cancers. They kill tumor cells in an antigen-specific MHC class II-restricted manner not only by cytolysis but also by release of IFNγ. Thus, CD4
CTL are interesting tools for cure approaches in chronic viral infections and cancer, but their potential to induce immunopathology has to be carefully taken into consideration.
Malignant cells express ligands for the natural killer cell immunoreceptor NKG2D, which sensitizes to early recognition and elimination by cytotoxic lymphocytes and provides an innate barrier against ...tumor development. However, the mechanisms that control NKG2D ligand (NKG2DL) expression in tumor cells remain unknown. We recently identified the NKG2DL ULBP2 as strong prognostic marker in human malignant melanoma. Here, we provide evidence that the tumor-suppressive microRNAs (miRNA) miR-34a and miR-34c control ULBP2 expression. Reporter gene analyses revealed that both miRNAs directly targeted the 3'-untranslated region of ULBP2 mRNA and that levels of miR-34a inversely correlated with expression of ULBP2 surface molecules. Accordingly, treatment of cancer cells with miRNA inhibitors led to upregulation of ULBP2, whereas miR-34 mimics led to downregulation of ULBP2, diminishing tumor cell recognition by NK cells. Treatment with the small molecule inhibitor Nutlin-3a also decreased ULBP2 levels in a p53-dependent manner, which was due to a p53-mediated increase in cellular miR-34 levels. Taken together, our study shows that tumor-suppressive miR-34a and miR-34c act as ULBP2 repressors. These findings also implicate p53 in ULBP2 regulation, emphasizing the role of the specific NKG2DL in tumor immune surveillance.
Myeloid derived suppressor cells (MDSCs) suppress innate and adaptive immunity, thereby limiting anti‐tumor immune responses in cancer patients. In patients with advanced melanoma, the phenotype and ...function of MDSCs remains controversial. In our study, we further explored two distinct subpopulations of MDSCs and investigated the impact of Vemurafenib on these cells. Flow cytometry analysis revealed that in comparison to healthy donors and patients with localized disease, PBMCs from patients with metastatic melanoma showed an increased frequency of CD14+HLA‐DR−/low monocytic MDSCs (moMDSCs) and of a previously unrecognized population of CD14−CD66b+Arginase1+ granulocytic MDSCs (grMDSCs). In vitro, both populations suppressed autologous T‐cell proliferation, which was tested in CFSE‐based proliferation assays. Vemurafenib treatment of melanoma patients reduced the frequency of both moMDSCs and grMDSCs. According to our in vivo finding, conditioned medium (CM) from Vemurafenib treated melanoma cells was less active in inducing moMDSCs in vitro than CM from untreated melanoma cells. In conclusion, patients with advanced melanoma show increased levels of moMDSCs, and of a population of CD14−CD66b+Arginase1+ grMDSCs. Both MDSCs are distinct populations capable of suppressing autologous T‐cell responses independently of each other. In vitro as well as in vivo, Vemurafenib inhibits the generation of human moMDSCs. Thus, Vemurafenib decreases immunosuppression in patients with advanced melanoma, indicating its potential as part of future immunotherapies.
What's new?
Censoring of the immune system, leading to its inability to mount an effective attack against tumor cells, is suspected to contribute to the advance of melanoma. The restrained response may be the result of two distinct populations of myeloid derived suppressor cells (MDSCs), as reported here. Monocytic MDSCs and a population of previously unrecognized Arginase1+ granulocytic MDSCs were detected at elevated frequencies in patients with metastatic melanoma. Frequencies of both subtypes declined in patients with clinical response to the enzyme inhibitor vemurafenib, which was further found to block in vitro generation of monocytic MDSCs.
Classical MHC molecules present processed peptides from endogenous protein antigens on the cell surface, which allows CD8
+
cytotoxic T lymphocytes (CTLs) to recognize and respond to the abnormal ...antigen repertoire of hazardous cells, including tumor cells. The light chain, β2-microglobulin (β2m), is an essential constant component of all trimeric MHC class I molecules. There is convincing evidence that β2m deficiency generates immune escape phenotypes in different tumor entities, with an exceptionally high frequency in colorectal carcinoma (CRC) and melanoma. Damage of a single
β2m
gene by LOH on chromosome 15 may be sufficient to generate a tumor cell precommitted to escape. In addition, this genetic lesion is followed in some tumors by a mutation of the second gene (point mutation or insertion/deletion), which produces a tumor cell unable to express any HLA class I molecule. The pattern of mutations found in microsatellite unstable colorectal carcinoma (MSI-H CRC) and melanoma showed a striking similarity, namely the predominance of frameshift mutations in repetitive CT elements. This review emphasizes common but also distinct molecular mechanisms of β2m loss in both tumor types. It also summarizes recent studies that point to an acquired β2m deficiency in response to cancer immunotherapy, a barrier to successful vaccination or adoptive cellular therapy.
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