Exploring immunotherapeutic targets in the tumor microenvironment

The immune system has developed along with the evolution of increasingly complex cellular organisms to sustain homeostasis and protect from threats. Cancer, a detrimental side effect of increasing organismic complexity, typically sequesters the immune system and hijacks its functions for its own prosperity. Cancer immunotherapy aims to harness the intrinsic potential of the immune system for the therapeutic benefit of cancer patients. The focus of this thesis is to identify and evaluate new immunotherapeutic targets in the tumor microenvironment, which can be modulated to restrict tumor growth and metastasis. Paper I describes a novel mechanism of interaction between marginal zone macrophages (MZMs) and marginal zone B cells (MZBs) in the spleen, which can be modulated by antibodies (Abs) to scavenger receptor MARCO on MZMs. This study demonstrates that MARCO targeting diminishes antigen (Ag) uptake by MZBs, which results in reduced Ag deposition in the splenic follicles. As anti-MARCO Abs can also be found in systemic lupus erythematosus (SLE), this interaction may affect subsequent adaptive immune responses to both self- and foreign antigen. Paper II identifies MARCO as a specific marker for a tumor-promoting macrophage subtype in the tumor microenvironment of mammary carcinoma, melanoma and colon carcinoma tumor models. Targeting MARCO on tumor-associated macrophages (TAMs) by Abs, inhibits tumor growth and metastasis and reprograms TAMs to an anti-tumor phenotype. Treatment with anti-MARCO mAbs in combination with checkpoint inhibitor, anti-CTLA-4 ab, may provide a promising approach for cancer immunotherary with clinical relevance for human breast cancer and melanoma. Paper III reveals the molecular mechanism through which tumor cells imitate immune cells, during epithelial-mesenchymal transition (EMT), prompting their targeted metastasis through the lymphatics. This study demonstrates that TGF-β, a known inducer of EMT, regulates the chemotactic axis CCR7/CCL21, directing preferential lymphatic dissemination of breast cancer cells. Paper IV evaluates dendritic cell-derived exosomes in a vaccination approach to reactivate adaptive anti-tumor responses. This study shows that dendritic cell-derived CD1d expressing exosomes loaded with α-Galactosylceramide (α-GalCer) can sensitize NKT cells and lead to subsequent activation of B cell and effector T cell responses, restricting tumor growth. In summary, the work presented in this thesis describes novel targets in the tumor microenvironment that can be used in immunotherapeutic approaches to re-activate endogenous mechanisms of innate and adaptive immunity against cancer. Additionally, it gives new insight into gene regulatory pathways controlling metastatic tumor spread, as well as utilizes custom designed biological molecules in anti-tumor vaccination strategies. Increasing our understanding of the intricate mechanisms regulating the immunosuppressive tumor microenvironment will reveal new knowledge and novel targets that can contribute to the design of prospective cancer immunotherapies

Successive crystal structure snapshots suggest the basis for MHC class i peptide loading and editing by tapasin

MHC-I epitope presentation to CD8 + T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptideloading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles

Elimination of reference mapping bias reveals robust immune related allele-specific expression in cross-bred sheep

Pervasive allelic variation at both gene and single nucleotide level (SNV) between individuals is commonly associated with complex traits in humans and animals. Allele-specific expression (ASE) analysis, using RNA-Seq, can provide a detailed annotation of allelic imbalance and infer the existence of cis-acting transcriptional regulation. However, variant detection in RNA-Seq data is compromised by biased mapping of reads to the reference DNA sequence. In this manuscript, we describe an unbiased standardized computational pipeline for allele-specific expression analysis using RNA-Seq data, which we have adapted and developed using tools available under open license. The analysis pipeline we present is designed to minimize reference bias while providing accurate profiling of allele-specific expression across tissues and cell types. Using this methodology, we were able to profile pervasive allelic imbalance across tissues and cell types, at both the gene and SNV level, in Texel×Scottish Blackface sheep, using the sheep gene expression atlas data set. ASE profiles were pervasive in each sheep and across all tissue types investigated. However, ASE profiles shared across tissues were limited, and instead, they tended to be highly tissue-specific. These tissue-specific ASE profiles may underlie the expression of economically important traits and could be utilized as weighted SNVs, for example, to improve the accuracy of genomic selection in breeding programs for sheep. An additional benefit of the pipeline is that it does not require parental genotypes and can therefore be applied to other RNA-Seq data sets for livestock, including those available on the Functional Annotation of Animal Genomes (FAANG) data portal. This study is the first global characterization of moderate to extreme ASE in tissues and cell types from sheep. We have applied a robust methodology for ASE profiling to provide both a novel analysis of the multi-dimensional sheep gene expression atlas data set and a foundation for identifying the regulatory and expressed elements of the genome that are driving complex traits in livestock

Effects of 1-MCP and storage temperature on quince fruit quality

Quince (Cydonia oblonga) is an important underutilized Mediterranean fruit having many possible culinary uses but there is very little knowledge on its storage and quality and almost nothing about 1-MCP effects on quince fruit quality. Quince fruit ('Afrata Volou') were harvested at commercial maturity (slightly green skin color), treated or not with 600 nl L-1 1-MCP five days later and stored for up to 151 d at 2°C (no cover) or 10°C (in loosely closed PE bags), or in a storage room with gradual (within a month) drop of storage temperature from 20 to 2°C (RH 80-90%). Control fruit were kept at ambient conditions (temperature 4 to 17°C and RH 35-95%). Quality evaluation included skin color, flesh firmness, juice soluble solids content (SSC) and titratable acidity and flesh dry matter (DM). Quince fruit skin color became more yellow, fruit softened, acidity and DM decreased and SSC only slightly decreased with time in storage. Fruit stored at 2°C or treated with 1-MCP remained greener based on lower a∗, b∗ and chroma values and higher hue angle values compared to fruit stored at higher temperatures. In contrast, storage at 2°C or treatment with 1-MCP did not improve quince flesh firmness. As expected, fruit kept at ambient conditions were the softest and also developed flesh browning. Quince fruit stored at 2°C or ambient conditions had lower SSC and DM values than fruit stored at 10°C in PE bags. It seems that low storage temperature or ambient temperature increase sugar losses through respiration and storing quince at 10°C in PE bags without treatment with 1-MCP is recommended to keep quince fruit quality acceptable for up to 106 d

Quality of fresh-cut apples treated at harvest with 1-MCP

Most apples diverted to long term storage are currently treated with 1-methylcyclopropane (1-MCP) in many countries. We tested the hypothesis that 1-MCP treatment could improve the quality of fresh-cut apple slices. 'Red Chief' and 'Granny Smith' apples treated or not with 600 nl L-1 of 1-MCP at harvest were sliced after 43 and 111 d ('Red Chief') or 33 and 90 d ('Granny Smith') of storage at 0°C, 90-95% RH. The slices were treated with water or 2% ascorbic acid + 1% CaCl2 (Asc+Ca), sealed in PE bags and stored at 1-2°C, 80-85% RH for three and six days. Freshly cut apple slices were used as reference control. Quality evaluation included flesh color and firmness and juice soluble solids content (SSC), acidity (TA) and total phenols. In both cultivars, slices from fruit treated with 1-MCP had similar color, SSC, TA and total phenol content and higher firmness than slices from fruit not treated with 1-MCP. In both cultivars, slice quality was similar after short or prolonged cold storage. The quality of slices after three days did not change significantly with three additional days of storage. Asc+Ca-treated slices usually had similar overall quality to freshly cut slices, but higher phenol content. Asc+Catreated slices had lighter and greener flesh color, higher total phenol content and similar firmness, SSC and TA to slices treated with water. In conclusion, 1-MCP treated apples gave harder fresh-cut product without any other quality differences compared with non-treated fruit, and treatment with 2% ascorbic acid + 1% CaCl2 successfully retained the overall quality of apple slices for at least six days

Alternative fertilization effects on peach and nectarine plant and fruit characteristics

Over- or under-fertilization is a common farmers’ practice in Greek peach orchards. During 2019 and 2020, 160% K were applied to ‘Onora’ peach and ‘Big Top’ nectarine mature trees, based on leaf and soil analyses, vegetative growth and expected yield, compared to farmer’s traditional practice (Control). Even though both cultivars were grown in the same orchard, soil properties were different with higher clay and pH in ‘Big Top’ compared to the ‘Onora’ planting area. All leaf macronutrients were at sufficiency levels and were not affected from the changes in fertilization practice. Soil surface temperature on tree row increased probably due to reduced vegetative growth and increased light penetration. Alternative fertilization had no effects on fruit size and yield of the two cultivars and differently affected fruit quality and antioxidant characteristics compared to control. Plant (Fv/Fm, crop water stress index, stem water potential) and leaf (specific leaf weight, chlorophyll, carotenoid and proline concentrations, relative water content, total phenols, antioxidant activity) response were differently affected from the alternative fertilization in each cultivar studied and each period of measurements (late June, early October). Reduced N and P and increased K fertilization differently affected fruit quality, and showed various positive or negative effects on plant and leaf characteristics studied. © 2022 International Society for Horticultural Science. All rights reserved

Nutrient inputs and outputs in commercial clingstone peach orchards in Greece based on conventional or sustainable fertilization practices

Clingstone peach production is important in Greece as large amounts of processed peaches are being exported throughout the world. Fertilization of peach farms is still conducted empirically with substantial amounts of N, P and K applied in late winter with basic composite fertilizers rarely followed by additional N and K or Ca applications in spring and summer. A more sustainable fertilization program was followed using <50% N, minor quantities of P, and similar or higher quantities of K than that applied with the local regime and with a different schedule compared to local practices in a number of farms and commonly grown cultivars. Inorganic nutrients were measured in fruits and prunings after two years of sustainable fertilization practices. Fruit yield and prunings volume were measured. The macronutrient inputs in the sustainable program were (in kg/ha): in 2019 <70 N, <30 P2O5, <100 K2O, and in 2020 <85 N, <40 P2O5, <155 K2O. Outputs with fruit normalized for 40 Mg ha-1 (most farms during the two years had lower yield) in 2020 were (in kg ha-1): <50 N, <11 P2O5, <4.3 CaO, <4.4 MgO, <80 K2O. Outputs with prunings in 2020 were (in kg ha-1): <25 N, <4 P2O5, <30 CaO, <3 MgO, <11 K2O. The balance between inputs and outputs was improved using the sustainable fertilization program, while fertilizer cost was variably affected and the possible environmental and economic burden due to nutrient losses decreased. © 2022 International Society for Horticultural Science. All rights reserved

Effects of foliar calcium applications and maturity stage on ‘Lemonato’ peach fruit quality and bruising severity

‘Lemonato’ peach is a traditional Greek series of clones particularly valued by the consumers because of its high fruit quality, flavor and aroma. The main problem of ‘Lemonato’ peach is flesh softening during ripening and the susceptibility of ripe fruit to bruising. Our hypothesis was that harvesting the fruit earlier than its commercial maturity could reduce fruit quality losses during postharvest handling. Secondly, innovative Ca formulations could reduce bruising incidence by strengthening cell walls and reducing flesh softening. In this work, two clones of ‘Lemonato’ peach (a mid-season ‘Andrea’ and a late-season ‘Mytera’) were used in order to study the effects of maturity stage, such as early harvest (EH) and commercial maturity (CM) on fruit quality. Moreover, the influence of foliar Ca applications on fruit quality with two formulations containing calcium-silicate in nanoparticles (CaSi) and organic Ca (OrCa) were also tested. Estimated fruit quality parameters included total phenols content (TPC), total antioxidant activity (TAA), and bruising severity. Compared to ‘Andrea’ clone, ‘Mytera’ fruit had better quality traits, higher TPC and TAA in both maturity stages, whereas at EH it had fruit mass similar to ‘Andrea’ and higher browning index (BI) fruit because of differences in tree fruit load. In all treatments and clones, CM fruit improved organoleptic characteristics compared to EH fruit. ‘Andreas’ fruit harvested at CM had higher fruit mass and significantly softer flesh than EH fruit without any effect from Ca applications. ‘Mytera’ fruit at CM had higher TPC and TAA than EH fruit in all treatments. Flesh browning caused by mechanical bruising increased with time mainly during the first 10 min after bruising in all treatments and maturity stages of both clones. Ca-treated fruit had slightly increased TPC and TAA, except of CM OrCa ‘Mytera’ fruit, which had lower quality parameters compared to control. Hovewer, this was not associated with BI. In both ‘Lemonato’ clones, Ca applications did not improve flesh firmness and BI at any maturity stage. © 2021 International Society for Horticultural Science. All rights reserved