Characterization of human γδ T lymphocytes as components of the early immune response

γδ T-Lymphozyten stellen eine numerisch kleine T-Zellsubpopulation dar, deren Funktionen Zytokinproduktion, Zytotoxizität, Antigenpräsentation, Produktion von AMPs, Erkennung von PAMPs durch Mustererkenungsrezeptoren und regulatorische Wirkung umfassen. Aufgrund der Verteilung von γδ T-Zellpopulationen in verschiedenen Geweben des Menschen war eine differentielle Chemokinrezeptorexpression zu vermuten und konnte in der vorliegenden Arbeit bestätigt werden. Dabei wurde die Expression von CCR4 als ein charakteristisches Merkmal für Vδ1 vs. Vδ2 γδ T-Zellen identifiziert. Desweiteren konnte ein differentielles Expressionsmuster von CCR6 und CCR9 mit den Lokalisationen von γδ T-Zellen aus Blut, Synovialflüssigkeit und Darmgewebe korreliert werden. Zur schnellen Erkennung von Pathogenen verfügen γδ T-Zellen neben dem T-Zellrezeptor auch über Mustererkennungsrezeptoren wie TLRs. Vorhergehende Studien hatten zudem erste Hinweise auf eine Expression des Mustererkennungsrezeptors NOD2 beschrieben. Die Expression des NOD2-Proteins in γδ T-Zellen konnte erstmalig in der vorliegenden Arbeit nachgewiesen werden. Weiterhin wurden über den TZR aktivierte γδ T-Zellen mit dem NOD2-Liganden MDP-LD im Vergleich mit dessen MDP-DD-Isomer stimuliert. Hochrein isolierte γδ T-Zellen zeigten eine verstärkte Freisetzung von CCL3/MIP-1α und IFN-γ, während in PBMZ befindliche γδ T-Zellen eine schwächere Proliferation nach Stimulation mit MDP-LD vs. MDP-DD aufwiesen. Nach Aktivierung setzen γδ T-Zellen Zytokine wie IFN-γ, TNF-α und CCL5/RANTES, aber auch antimikrobielle Peptide wie LL-37 frei. In der vorliegenden Arbeit wurden γδ T-Zellen mit dem Überstand adhärent wachsender Ps. aeruginosa stimuliert um die nachfolgende Expression von Zytokinen und AMPs zu untersuchen. Dabei konnte eine induzierbare Expression der Zytokine IFN-γ und CCL20/MIP-3α festgestellt werden. Eine ebenfalls stimulierbare antimikrobielle Aktivität von γδ T-Zellen ließ sich z.T. auf die Produktion und/oder Freisetzung von Granulysin, Granzym B, HBD-1 und Elafin zurückführen. In der vorliegenden Arbeit wurden Aspekte von γδ T-Lymphozyten im Hinblick auf ihre Lokalisation und die Erkennung und Bekämpfung von Pathogenen näher untersucht. Die vorliegenden Ergebnisse haben somit einen Beitrag zu einem tieferen Verständnis der Bedeutung von humanen γδ T-Zellen in der frühen Immunantwort erbracht.γδ T cells represent a numerical small T cell subset displaying functional activity including cytokine production, cytotoxicity, antigen presentation, production of antimicrobial peptides, recognition of PAMPs by pattern recognition receptors and regulatory functions. According to the distribution of γδ T cell subsets in different human tissues, a differential expression of chemokine receptors was expected and could be confirmed in the present study. The expression of CCR4 was identified as a characteristic feature of Vδ1 vs. Vδ2 γδ T cells. Furthermore, a differential expression pattern of CCR6 and CCR9 was correlated with the localizations of γδ T cells from blood, synovial fluid and intestine. For rapid recognition of pathogens, γδ T cells are equipped with not just the T cell receptor, but with pattern recognition receptors including TLRs. Additionally, previous studies had described preliminary evidences for an expression of the pattern recognition receptor NOD2. In the present study, the expression of the NOD2-protein was described for the first time. Furthermore, T cell receptor activated γδ T cells were treated with the NOD2-ligand MDP-LD in comparison to the MDP-DD-isomer. Stimulated with MDP-LD vs. MDP-DD, highly purified γδ T cells showed a stronger release of CCL3/MIP-1α and IFN-γ, while γδ T cells within peripheral mononuclear blood cells exhibited an attenuated proliferation. After activation, γδ T cells release cytokines such as IFN-γ, TNF-α and CCL5/RANTES as well as antimicrobial peptides like LL-37. In the present study, γδ T cells were stimulated by the supernatant of adherent Ps. aeruginosa in order to investigate the consecutive expression of cytokines and antimicrobial peptides. An inducible expression of the cytokines IFN-γ and CCL20/MIP-3α was observed. The similarly inducible antimicrobial activity of γδ T cells was attributed partly to the production and/or release of granulysin, granzyme B, human β-defensin 1 and elafin. In the present study, aspects of γδ T lymphocytes concerning their localization and the recognition of and the defense against pathogens were examined. The presented results have contributed to a deeper insight into the early immune response of human γδ T cells

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer

Human NK cells adapt their immune response towards increasing multiplicities of infection of Aspergillus fumigatus

Background: The saprophytic fungus Aspergillus fumigatus reproduces by generation of conidia, which are spread by airflow throughout nature. Since humans are inhaling certain amounts of spores every day, the (innate) immune system is constantly challenged. Even though macrophages and neutrophils carry the main burden, also NK cells are regarded to contribute to the antifungal immune response. While NK cells reveal a low frequency, expression and release of immunomodulatory molecules seem to be a natural way of their involvement. Results: In this study we show, that NK cells secrete chemokines such as CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES early on after stimulation with Aspergillus fumigatus and, in addition, adjust the concentration of chemokines released to the multiplicity of infection of Aspergillus fumigatus. Conclusions: These results further corroborate the relevance of NK cells within the antifungal immune response, which is regarded to be more and more important in the development and outcome of invasive aspergillosis in immunocompromised patients after hematopoietic stem cell transplantation. Additionally, the correlation between the multiplicity of infection and the expression and release of chemokines shown here may be useful in further studies for the quantification and/or surveillance of the NK cell involvement in antifungal immune responses

Reconstituting NK cells after allogeneic stem cell transplantation show impaired response to the fungal pathogen Aspergillus fumigatus

Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56brightCD16dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility

Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections