Scandinavian Journal of Immunology最新文献

Evaluating immune status is a challenging and time-consuming process that involves analysing various biomarkers through numerous assays. The sensitive label-free technique of multispectral imaging of cell autofluorescence involves directly assessing the molecular composition of cells to gather biological information. Cells were cultured in RPMI 1640 modified media supplemented with penicillin-streptomycin and 10% foetal bovine serum at 37°C, with 5% CO₂ and 95% humidity. Activation and differentiation was confirmed using immunofluorophores against relevant markers. Multispectral microscopy utilized defined spectral regions, which spanned the excitation (345-476 nm) and emission (414-675 nm) wavelength ranges. In total, 56 distinct spectral channels were applied. These channels cover the spectrum of several fluorophores notably NAD(P)H and flavins, whose concentrations depend on cellular metabolism. We identified distinct spectral signatures for characterizing cells from the Jurkat, Ramos, THP-1, and HL-60 immune cell lines. These signatures correspond to four major immune cell types: T cells (Lymphocytes), B cells (Lymphocytes), monocytes and neutrophils. Moreover, our investigation explored the potential identification of both activated and resting forms of these cells, including the discrimination of M0, M1 and M2 polarized macrophages. Classification accuracy ranged from 92% to 100% based on receiver operator characteristic area under the curve (ROC AUC) assessment. These results indicate that the multispectral evaluation of cell autofluorescence is applicable for characterization of immune status. This includes the assessment of cell types and their activation status, all achievable through a single non-invasive assay.

MAIT cells are one of the largest unconventional T cell populations and, recruited to the site of infection, play both protective and pathogenic roles during pulmonary viral infections. MAIT cell activation patterns change significantly during COVID-19, with a notable decrease in their frequency in peripheral blood of severe cases. In the present study, we aimed to investigate the expression profiles of various immune checkpoint pathways on MAIT, MAIT-like and non-MAIT cells in moderate and severe COVID-19 patients undergoing cytokine storm. Despite numerous studies comparing MAIT cell characteristics based on COVID-19 disease severity, none have delved into the critical differences in MAIT cell immune checkpoint profiles between moderate and severe COVID-19 patients, all experiencing a cytokine storm. Flow cytometry was used to analyse peripheral blood mononuclear cells from a cohort of 35 patients, comprising 18 moderate and 17 severe cases, alongside 14 healthy controls. Our investigation specifically focuses on severe COVID-19 presentations, revealing a marked deletion of MAIT cells. Further exploration into the regulatory dynamics of MAIT cell functionality reveals shifts in the expression profiles of critical immune checkpoint receptors, notably PD-1 and CD226. In severe COVID-19 patients, MAIT cells showed a significant decrease in the expression of CD226, whereas MAIT-like and non-MAIT cells demonstrated a significant increase in the expression of PD-1 compared to healthy individuals. The expression of the TIGIT receptor remained unaltered across all investigated groups. Our findings contribute to the existing knowledge by elucidating the changes in MAIT cell subpopulations and their potential role in COVID-19 disease severity.

Oocyte donation (OD) pregnancies show a higher fetal-maternal incompatibility and a higher risk of developing pre-eclampsia (PE) than autologous pregnancies. As maternal monocytes play a role in the tolerization of the allogeneic fetus, the aim of this study was to analyse monocyte phenotypes in healthy and PE OD pregnancies. We collected maternal peripheral blood at different gestational time points in healthy (n = 10) and PE (n = 5) OD pregnancies. Fetal-maternal human leukocyte antigen (HLA) mismatches were calculated. We used a 35-colour antibody panel for Aurora spectral flow cytometry to analyse the composition and surface marker expression of monocyte subsets. Expression of CD38 on intermediate monocytes significantly increased throughout gestation in healthy OD pregnancies. Compared with the healthy group, the PE group exhibited even higher CD38 expression on monocyte subsets, with statistical significance. Immune inhibiting receptors CD85j (LILRB1) and CD85d (LILRB2), as well as monocyte recruitment regulating molecules CCR2 and CD91, also showed significantly enhanced expression on monocyte subsets during PE. When comparing healthy and PE OD only in pregnancies with high HLA mismatches, the different CD38 and CD85j expression in monocyte subsets was still significant. In conclusion, in healthy OD pregnancies, the upregulated CD38 expression might reflect a proinflammatory condition specifically at the third trimester. In PE OD pregnancies, expression of both inflammatory and immune regulatory markers is increased in maternal peripheral monocyte subsets. The elevated expression of CCR2 and CD91 on these subsets might reflect monocyte chemotaxis and the effect from systemic vascular dysfunction at the late stage of PE.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells playing a critical role in immune suppression. In vitro-generated MDSCs are a convenient tool to study the properties of tumour-associated MDSCs. Here, we compared six protocols for in vitro generation of functional mouse MDSCs from bone marrow progenitors. The protocols included granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G-CSF), with or without a tumour-conditioned medium (TCM) derived from B16-F10 melanoma. Obtained MDSCs were characterized by morphology, phenotype, gene expression of key immunosuppressive factors, and in vitro suppression of T cell proliferation. All tested protocols yielded approximately 25% monocytic and 50% polymorphonuclear MDSCs. Protocols using IL-6 generated MDSCs with reduced maturation and differentiation status, upregulated Arg1 and Nos1 mRNA expression, increased levels of Arg-1 and TGF-β proteins and enhanced ROS production compared to the other protocols. All tested protocols yielded MDSCs that efficiently inhibited T cell proliferation in vitro, with some advantage for the GM-CSF and G-CSF + GM-CSF protocols. Interestingly, a combination of protocols with B16-F10-derived TCM resulted in the generation of MDSCs with reduced immunosuppressive properties. Our results provide valuable insights into the optimal conditions for in vitro generation of MDSCs with specific immunosuppressive properties.

The group of patients with DNA-repair-defects increases susceptibility to infections due to impaired repertoire diversity. In this context, we aimed to investigate the TCRvβ-repertoire by flow cytometric analysis and its correlation with clinical entities in a group of IEI patients with DNA repair defects. Peripheral lymphocyte subset and TCRvβ-repertoire analyses were performed by flow cytometric analysis. The aim was to explore the changing TCR-Vβ-repertoire that can predict some clinical entities by investigating the repertoire using flow-cytometric-analysis-based TCR-Vβ and its interaction with clinical entities in a group of IEI patients with DNA repair defects. TCR-repertoire of the patients with DNA-repair-defects and healthy controls was analysed with flow-cytometer. The potential of flow-cytometric analysis of the TCR repertoire as a practical and easily accessible clinical prediction method was investigated. Thirty-nine-IEI patients with DNA-repair-defects and 15 age-matched healthy-controls were included in this study. Peripheral lymphocyte subset and TCR-Vβ repertoire analyses were performed by flow cytometry. Compared to the control group, 9 out of 24 clones (37.5%) exhibited a statistically significant reduction, while only 3 clones showed a statistically significant increase (p < 0.05). Preferential use of vβ-genes was associated with some clinical entities. Lower TCR-vβ-9 and TCR-vβ23, higher TCR-vβ7.2 were found in the patients with pneumonia (n = 13) (p = 0.018, p = 0.044 p = 0.032). AT patients with pneumonia had lower TCR-vβ-9 clone than patients without pneumonia (p = 0.008). Skewed proliferation of most TCR-vβ clones was seen DNA-repair-defects, especially AT. In addition, this study showed that preferential use of TCR-vβ genes could be predictive for some clinical entities.

The effects of vitamin D and vitamin A in immune cells are mediated through the vitamin D receptor (VDR) and retinoic acid receptor (RAR), respectively. These receptors share the retinoid X receptor (RXR) co-factor for transcriptional regulation. We investigated the effects of active vitamin D₃ (1,25(OH)₂D₃) and 9-cis retinoic acid (9cRA) on T helper (T_H)1 and T_H2 cytokines and transcription factors in primary human blood-derived CD4⁺ T cells. We aimed to address the discrepancies in this field, particularly regarding the effects of 9cRA and the vitamins in combination. 1,25(OH)₂D₃ upregulated IL-13 and suppressed IFNγ, while 9cRA had the opposite effects. This was largely independent of a T_H1/T_H2 phenotype shift. Combined vitamin supplementation produced intermediate cytokine levels, not only through transcriptional regulation by VDR-RXR and RAR-RXR but also through 1,25(OH)₂D₃ counteracting the effects of 9cRA on solely 9cRA-responsive genes. Similar results were observed in hereditary vitamin D-resistant rickets (HVDRR) patient T cells, where VDR cannot bind to DNA, indicating that RXR binding to either receptor can limit the other's activity. Additionally, we observed downregulated RAR upon 9cRA supplementation and its re-localization out of the nucleus upon 1,25(OH)₂D₃ supplementation, suggesting a mechanism of indirect regulation by VDR. VDR protein levels were also upregulated upon 9cRA supplementation, suggesting a novel negative feedback mechanism of 9cRA transcriptional activity, whereby 9cRA promotes its own competitor. This study sets the stage for future research into the combined immunomodulatory mechanisms of 1,25(OH)₂D₃ and 9cRA, involving both direct transcriptional regulation and indirect regulation via RXR competitive binding.

Dry eye disease (DED) is an inflammatory disorder in which CD4⁺ T cells play a significant role in its pathogenesis. A CD4⁺ T cell subset termed granulocyte-macrophage colony-stimulating factor-producing T helper (ThGM) cells would contribute to DED pathogenesis. However, the mechanisms by which the activity of ThGM cells is modulated are not thoroughly understood. In this research, we characterized the effects of neurokinin 1 receptor (NK1R) and neurokinin 2 receptor (NK2R) on ThGM cells and T helper 1 (Th1) cells in a murine DED model. We found that ThGM cells expressed NK1R and NK2R, whereas Th1 cells predominantly expressed NK1R. Furthermore, substance P and neurokinin A (NKA), the ligands of NK1R and NK2R, were upregulated in post-DED LNs and conjunctivae. Substance P significantly promoted granulocyte-macrophage colony-stimulating factor (GM-CSF) expression while mildly upregulating the expression of interferon-gamma (IFN-γ) and interleukin 2 (IL-2) in ThGM cells. By contrast, NKA did not change GM-CSF expression but significantly increased IFN-γ expression in ThGM cells. Importantly, the adoptive transfer of NK1R-expressing ThGM cells significantly exacerbated DED, whereas the transfer of NK1R-knockdown ThGM cells weakly aggravated DED. NK2R knockdown in ThGM cells did not affect DED progression. In conclusion, this study identifies the substance P-NK1R axis as a novel mechanism that reinforces the pathogenicity of ThGM cells in DED.