Distinct Mechanisms of Tumor Resistance to NK Killing: Of Mice and Men

Tumors cells can release natural killer (NK) cell ligands for activating receptor NKG2D that are thought to inhibit NK cell function. In a recent issue of Science, Deng et al. (2015) show that, unexpectedly, a soluble NKG2D ligand can enhance anti-tumor NK cell activity

In Vivo Cross-Priming of MHC Class I–Restricted Antigens Requires the TAP Transporter

AbstractRecent in vitro evidence suggests two alternative mechanisms by which bone marrow–derived APCs may process exogenous antigens for presentation to CTL in vivo, a phenomenon termed cross-priming. Although in vitro studies have suggested that both TAP-dependent and TAP-independent pathways exist, we have now demonstrated an absolute requirement for a functional TAP for cross-priming to occur in vivo. Bone marrow chimeras reconstituted with marrow from TAP-defective donors develop functional CD8+ CTL, but have APCs with disrupted TAP function. In such chimeras, in vivo priming of naive CTL was observed when antigen was targeted to the ER in a TAP-independent fashion, but cross-priming could not be demonstrated. These results support the TAP-dependent mechanism of cross-priming

Slow-Release Formulation of Cowpea Mosaic Virus for In Situ Vaccine Delivery to Treat Ovarian Cancer.

The plant viral nanoparticle cowpea mosaic virus (CPMV) is shown to be an effective immunotherapy for ovarian cancer when administered as in situ vaccine weekly, directly into the intraperitoneal (IP) space in mice with disseminated tumors. While the antitumor efficacy is promising, the required frequency of administration may pose challenges for clinical implementation. To overcome this, a slow release formulation is developed. CPMV and polyamidoamine generation 4 dendrimer form aggregates (CPMV-G4) based on electrostatic interactions and as a function of salt concentration, allowing for tailoring of aggregate size and release of CPMV. The antitumor efficacy of a single administration of CPMV-G4 is compared to weekly administration of soluble CPMV in a mouse model of peritoneal ovarian cancer and found to be as effective at reducing disease burden as more frequent administrations of soluble CPMV; a single injection of soluble CPMV, does not significantly slow cancer development. The ability of CPMV-G4 to control tumor growth following a single injection is likely due to the continued presence of CPMV in the IP space leading to prolonged immune stimulation. This enhanced retention of CPMV and its antitumor efficacy demonstrates the potential for viral-dendrimer hybrids to be used for delayed release applications

Are the IL-2 Receptors Expressed in the Murine Fetal Thymus Functional?

It is well established that the majority of murine fetal thymocytes (day 15 of gestation) express receptors for interleukin 2 (IL-2), but the functional significance of these IL-2 receptors (IL-2Rs) is not clear. In situ hybridization data show a developmentally regulated expression of IL-2 and IL-2R mRNA. IL-2 binding studies were performed on fetal thymocytes and the results show the presence of both high (kD ≅ 20 pM) and low (kD ≅ 10 nM) affinity IL-2Rs. These IL-2Rs are indeed functional: intact fetal thymic lobes (but not cell suspensions) cultured in IL-2 exhibited an in vitro proliferative response at 20 pM of IL-2, corresponding with the presence of a functional high-affinity IL-2R on fetal thymocytes. The IL-2-dependent growth was primarily observed in the IL-2R + thymic subset, which contains the CD3-/CD4-/CD8- precursor thymocytes. Furthermore, in vitro blocking of IL-2 in intact fetal thymic lobes resulted in a reduction in the cell yield, which predominantly affected the expansion of the immature CD3-/CD4-/CD8-thymocytes. Our findings strongly support the concept that the IL-2/IL-2R pathway is responsible for the proliferation of precursor cells within the fetal thymus

Partially circumventing peripheral tolerance for oncogene-specific prostate cancer immunotherapy

BACKGROUND Failure of cancer immunotherapy is essentially due to immunological tolerance to tumor-associated antigens (TAAs), as these antigens are also expressed in healthy tissues. METHODS Here, we used transgenic adenocarcinoma of mouse prostate (TRAMP) mice, which develop lethal prostate cancer due to prostate-specific expression of SV40 T antigen (Tag), to evaluate effects of prostatic transformation on oncogene TAA-specific tolerance and to test the possibility of breaking such tolerance using a modified recombinant vaccinia virus. RESULTS We showed that Tag expression in TRAMP mice is uniquely extra-thymic, and levels of prostatic Tag expression positively correlate with malignant transformation of the prostate. Yet, young tumor-free TRAMP mice were tolerant to Tag antigen. We therefore attempted overcoming such peripheral oncogene-specific T cell tolerance through immunization with a vaccinia construct encoding Tag immunogenic epitopes. This vaccination modality showed that oncogene-specific tolerance was successfully overcome by effective in vivo priming of Tag-specific cytotoxic T cells (CTLs). However, this was restricted to young TRAMP mice. Tag-specific CTL from “tumor naÏve” young TRAMP mice showed significant anti-tumor efficacy in vivo by eliminating established heterotopic prostate tumors and prolonging survival in SCID mice harboring Tag-expressing tumors. In contrast, older TRAMP mice with established prostate tumors exhibited oncogene-specific tolerance as evidenced by failure to generate Tag-specific CTL following Tag-specific immunization. CONCLUSIONS Peripheral tolerance can be overcome for effective anti-tumor therapy following oncogene-specific immunization. However, this ability to elicit oncogene-specific CTL is impeded in the tumor-bearing host, in the context of increased oncogene expression associated with tumor progression. Prostate 68: 715–727, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58560/1/20689_ftp.pd

Enhanced Antigen-Specific Antitumor Immunity with Altered Peptide Ligands that Stabilize the MHC-Peptide-TCR Complex

AbstractT cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I–restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells

Alterations of immune response of non-small lung cancer with azacytidine

Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA - Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints - in particular the PD-1/PD-L1 pathway - may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with 3H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed