Surface protein and functional analyses identify CD4+CD39+ TCR αß+ and activated TCR Vδ1+ cells with distinct pro-inflammatory functions in Crohn's disease lesions.

Crohn's disease (CD) is a chronic immune-mediated disorder of the gastrointestinal tract. Extensive screening studies have revealed the accumulation of immune cell subsets with unique plasticity and immunoregulatory properties in patients with CD. We performed phenotypic and functional studies on inflamed and non-inflamed bioptic tissue to investigate the presence of distinct T cells in the intestinal mucosa of CD patients. We analysed hundreds of surface molecules expressed on cells isolated from the intestinal tissue of CD patients using anti-CD45 mAbs-based barcoding. A gene ontology enrichment analysis showed that proteins that regulate the activation of T cells were the most enriched group. We, therefore, designed T-cell focused multicolour flow-cytometry panels and performed clustering analysis which revealed an accumulation of activated TEM CD4+CD39+ T cells producing IL-17 and IL-21 and increased frequency of terminally differentiated TCR Vδ1+ cells producing TNF-α and IFN-γ in inflamed tissue of CD patients. The different functional capacities of CD4+ and TCR Vδ1+ cells in CD lesions indicate their non-overlapping contribution to inflammation. The abnormally high number of terminally differentiated TCR Vδ1+ cells suggests that they are continuously activated in inflamed tissue, making them a potential target for novel therapies.

miR-150 downregulation contributes to the high-grade transformation of follicular lymphoma by upregulating FOXP1 levels.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Impacts of priming on distinct immunosuppressive mechanisms of mesenchymal stromal cells under translationally relevant conditions.

BACKGROUND: The multimodal properties of mesenchymal stromal cells (MSCs), particularly their ability to modulate immune responses is of high interest in translational research. Pro-inflammatory, hypoxic, and 3D culture priming are promising and often used strategies to improve the immunosuppressive potency of MSCs, but the underlying mechanisms are not well understood. Therefore, the aims of this study were (i) to compare the effects of pro-inflammatory, hypoxic, and 3D culture priming on the in vitro immunosuppressive potential of MSCs, (ii) to assess if immunosuppressive priming effects are temporally preserved under standard and translationally relevant culture conditions, and (iii) to investigate if the three priming strategies engage the same immunosuppressive mechanisms. METHODS: Functional in vitro T cell suppressive potency measurements were conducted to assess the impact of pro-inflammatory, hypoxic, and 3D culture priming on the immunosuppressive potential of human bone marrow-derived MSCs. Primed MSCs were either cultured under standard cell culture conditions or translationally relevant culture conditions, and their transcriptomic adaptations were monitored over time. Next-generation sequencing was performed to assess if different priming strategies activate distinct immunosuppressive mechanisms. RESULTS: (i) Pro-inflammatory, hypoxic, and 3D culture priming induced profound transcriptomic changes in MSCs resulting in a significantly enhanced T cell suppressive potential of pro-inflammatory and 3D culture primed MSCs. (ii) Priming effects rapidly faded under standard cell culture conditions but were partially preserved under translationally relevant conditions. Interestingly, continuous 3D culture priming of MSCs maintained the immunosuppressive potency of MSCs. (iii) Next-generation sequencing revealed that priming strategy-specific differentially expressed genes are involved in the T cell suppressive capacity of MSCs, indicating that different priming strategies engage distinct immunosuppressive mechanisms. CONCLUSION: Priming can be a useful approach to improve the immunosuppressive potency of MSCs. However, future studies involving primed MSCs should carefully consider the significant impact of translationally relevant conditions on the preservation of priming effects. Continuous 3D culture could act as a functionalized formulation, supporting the administration of MSC spheroids for a sustainably improved immunosuppressive potency.

Intervertebral disc microbiome in Modic changes: Lack of result replication underscores the need for a consensus in low-biomass microbiome analysis.

Introduction: The emerging field of the disc microbiome challenges traditional views of disc sterility, which opens new avenues for novel clinical insights. However, the lack of methodological consensus in disc microbiome studies introduces discrepancies. The aims of this study were to (1) compare the disc microbiome of non-Modic (nonMC), Modic type 1 change (MC1), and MC2 discs to findings from prior disc microbiome studies, and (2) investigate if discrepancies to prior studies can be explained with bioinformatic variations. Methods: Sequencing of 16S rRNA in 70 discs (24 nonMC, 25 MC1, and 21 MC2) for microbiome profiling. The experimental setup included buffer contamination controls and was performed under aseptic conditions. Methodology and results were contrasted with previous disc microbiome studies. Critical bioinformatic steps that were different in our best-practice approach and previous disc microbiome studies (taxonomic lineage assignment, prevalence cut-off) were varied and their effect on results were compared. Results: There was limited overlap of results with a previous study on MC disc microbiome. No bacterial genera were shared using the same bioinformatic parameters. Taxonomic lineage assignment using "amplicon sequencing variants" was more sensitive and detected 48 genera compared to 22 with "operational taxonomic units" (previous study). Increasing filter cut-off from 4% to 50% (previous study) reduced genera from 48 to 4 genera. Despite these differences, both studies observed dysbiosis with an increased abundance of gram-negative bacteria in MC discs as well as a lower beta-diversity. Cutibacterium was persistently detected in all groups independent of the bioinformatic approach, emphasizing its prevalence. Conclusion: There is dysbiosis in MC discs. Bioinformatic parameters impact results yet cannot explain the different findings from this and a previous study. Therefore, discrepancies are likely caused by different sample preparations or true biologic differences. Harmonized protocols are required to advance understanding of the disc microbiome and its clinical implications.

Bone marrow stromal cells in Modic type 1 changes promote neurite outgrowth.

The pain in patients with Modic type 1 changes (MC1) is often due to vertebral body endplate pain, which is linked to abnormal neurite outgrowth in the vertebral body and adjacent endplate. The aim of this study was to understand the role of MC1 bone marrow stromal cells (BMSCs) in neurite outgrowth. BMSCs can produce neurotrophic factors, which have been shown to be pro-fibrotic in MC1, and expand in the perivascular space where sensory vertebral nerves are located. The study involved the exploration of the BMSC transcriptome in MC1, co-culture of MC1 BMSCs with the neuroblastoma cell line SH-SY5Y, analysis of supernatant cytokines, and analysis of gene expression changes in co-cultured SH-SY5Y. Transcriptomic analysis revealed upregulated brain-derived neurotrophic factor (BDNF) signaling-related pathways. Co-cultures of MC1 BMSCs with SH-SY5Y cells resulted in increased neurite sprouting compared to co-cultures with control BMSCs. The concentration of BDNF and other cytokines supporting neuron growth was increased in MC1 vs. control BMSC co-culture supernatants. Taken together, these findings show that MC1 BMSCs provide strong pro-neurotrophic cues to nearby neurons and could be a relevant disease-modifying treatment target.

New concepts in follicular lymphoma biology: From BCL2 to epigenetic regulators and non-coding RNAs.

The molecular pathogenesis of follicular lymphoma (FL) was partially revealed 3 decades ago, with the discovery of the translocation that brings BCL2 under the influence of immunoglobulin heavy chain enhancers in a vast majority of cases. Despite the importance of this seminal observation, it has become increasingly clear that additional genetic alterations need to occur to trigger neoplastic transformation and disease progression. The evolution of FL involves developmental arrest and disruption of the normal function of one or more of epigenetic regulators including KMT2D/MLL2, EZH2, CBP/CREBBP, p300/EP300, and HIST1H1 in >95% of cases. B-cells "arrested" in germinal centers acquire dozens of additional genetic aberrations that influence key pathways controlling their physiological development including B Cell Receptor (BCR) signaling, PI3K/AKT, TLR, mTOR, NF-κB, JAK/STAT, MAPK, CD40/CD40L, chemokine, and interleukin signaling. Additionally, most cases of FL do not result from linear accumulation of genomic aberrations, but rather evolve from a common progenitor cell population by diverse evolution, creating multiple FL subclones in one patient. Moreover, one of the subclones might acquire a combination of aberrations involving genes controlling cell survival and proliferation including MDM2, CDKN2A/B, BCL6, MYC, TP53, ß2M, FOXO1, MYD88, STAT3, or miR-17-92, and this can lead to the transformation of an initially indolent FL to an aggressive lymphoma (2%-3% risk per year). The complexity of the disease is also underscored by the importance of its interactions with the microenvironment that can substantially influence disease development and prognosis. Interpreting individual aberrations in relation to their impact on normal processes, their frequency, position in the disease evolution, and the consequences of their (co)occurrence, are the basis for understanding FL pathogenesis. This is necessary for the identification of patients with risk of early progression or transformation, for the development of novel targeted therapies, and for personalized treatment approaches. In this review, we summarize recent knowledge of molecular pathways and microenvironmental components involved in FL biology, and discuss them in the context of physiological B-cell development, FL evolution, and targeted therapies.