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[Introduction]: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. [Methods]: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. [Results]: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. [Conclusion]: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

[Introduction]: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease., [Methods]: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells., [Results]: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines., [Conclusion]: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

In recent decades, the demand for biomedical imaging tools has grown very rapidly as a key feature for biomedical research and diagnostic applications. Particularly, fluorescence imaging has gained increased attention as a non-invasive, inexpensive technique that allows real-time imaging. However, tissue auto-fluorescence under external illumination, together with a weak tissue penetration of low wavelength excitation light, largely restricts the application of the technique. Accordingly, new types of fluorescent labels are currently being investigated and, in this search, phosphorescent nanoparticles promise great potential, as they combine the interesting size-dependent properties of nanoscale materials with a long-lasting phosphorescence-type emission that allows optical imaging well after excitation (so avoiding autofluorescence). In this work, core-shell structures consisting of SrAlO:Eu,Dy luminescent cores encapsulated within a biocompatible silica shell were prepared, showing a green persistent phosphorescence with an afterglow time of more than 1000 s. A high-energy ball milling procedure was used to reduce the size of the starting phosphors to a size suitable for cellular uptake, while the silica coating was produced by a reverse micelle methodology that eventually allows the excitation and emission light to pass efficiently through the shell. Confocal fluorescence microscopy using HeLa cancer cells confirmed the potential of the all-ceramic composites produced as feasible labels for in vitro optical imaging.

Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof., [ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof., [ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof., [ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof., [ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.

Bacterial phagocytosis and antigen cross-presentation to activate CD8 T cells are principal functions of professional antigen presenting cells. However, conventional CD4 T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 T cells, which proliferate and become cytotoxic in response. CD4 T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 T cells with low PD-1 expression. Moreover, transphagocytic CD4 T cells induce protective anti-tumour immune responses by priming CD8 T cells, highlighting the potential of CD4 T cells as a tool for cancer immunotherapy.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.

Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69–/– DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69–/– DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P1) by CD69–/– DCs, whereas S1P3 expression levels were similar in wild-type and CD69–/– DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P1 function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery