Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

Dual targeting negative enrichment strategy for highly sensitive and purity detection of CTCs.

Circulating tumor cells (CTCs) have significant clinical value in early tumor detection, dynamic monitoring and immunotherapy. CTC detection stands out as a leading non-invasive approach for tumor diagnostics and therapeutics. However, the high heterogeneity of CTCs and the occurrence of epithelial-mesenchymal transition (EMT) during metastasis pose challenges to methods relying on EpCAM-positive enrichment. To address these limitations, a method based on negative enrichment of CTCs using specific leukocyte targets has been developed. In this study, aiming to overcome the low purity associated with immunomagnetic beads targeting solely the leukocyte common antigen CD45, we introduced CD66b-modified immunomagnetic beads. CD66b, a specific target for neutrophils with abundant residues, was chosen as a complementary approach. The process involved initial collection of nucleated cells from whole blood samples using density gradient centrifugation. Subsequently, magnetically labeled leukocytes were removed by magnetic field, enabling the capture of CTCs with higher sensitivity and purity while retaining their activity. Finally, we selected 20 clinical blood samples from patients with various cancers to validate the effectiveness of this strategy, providing a new generalized tool for the clinical detection of CTCs.

Memory T-cell enriched haploidentical transplantation with NK cell addback results in promising long-term outcomes: a phase II trial.

BACKGROUND: Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen. METHODS: In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy. RESULTS: The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72-98) and 88% (95% CI: 67-96); ≥ CR2 were 81% (95% CI: 61-92) and 68% (95% CI: 47-82) and refractory disease were 32% (95% CI: 11-54) and 20% (95% CI: 6-40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64-87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/µL and 140/µL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035). CONCLUSION: The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611).

Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) as a CD45 Protein Stability Regulator.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a binding partner of the phosphatase CD45, but its function remains poorly understood. Its close interaction with CD45 suggests that LPAP may potentially regulate CD45, but direct biochemical evidence for this has not yet been obtained. We found that in the Jurkat lymphoid cells the levels of LPAP and CD45 proteins are interrelated and well correlated with each other. Knockout of LPAP leads to the decrease in the surface expression of CD45, while its overexpression, on the contrary, caused its increase. No such correlation was found in the non-lymphoid K562 cells. We hypothesize that LPAP regulates expression level of CD45 and thus can affect lymphocyte activation.

The VISTA/VSIG3/PSGL-1 axis: crosstalk between immune effector cells and cancer cells in invasive ductal breast carcinoma.

A checkpoint protein called the V-domain Ig suppressor of T cell activation (VISTA) is important for controlling immune responses. Immune cells that interact with VISTA have molecules, or receptors, known as VISTA receptors. Immune system activity can be modified by the interaction between VISTA and its receptors. Since targeting VISTA or its receptors may be beneficial in certain conditions, VISTA has been studied in relation to immunotherapy for cancer and autoimmune illnesses. The purpose of this study was to examine the expression levels and interactions between VISTA and its receptors, VSIG3 and PSGL-1, in breast cancer tissues. IHC analysis revealed higher levels of proteins within the VISTA/VSIG3/PSGL-1 axis in cancer tissues than in the reference samples (mastopathies). VISTA was found in breast cancer cells and intratumoral immune cells, with membranous and cytoplasmic staining patterns. VISTA was also linked with pathological grade and VSIG3 and PSGL-1 levels. Furthermore, we discovered that the knockdown of one axis member boosted the expression of the other partners. This highlights the significance of VISTA/VSIG3/PSGL-1 in tumor stroma and microenvironment remodeling. Our findings indicate the importance of the VISTA/VSIG3/PSGL-1 axis in the molecular biology of cancer cells and the immune microenvironment.

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis.

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5'-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchuslabrax.

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

High sodium, rather than high blood pressure, induces immune cell activation and renal infiltration in ovariectomized adult Wistar rats.

We used an animal model of salt-sensitive hypertension (SSH) in which ovariectomized (oVx) rats developed hypertension with high salt (HS) intake. Hypertension is accompanied by changes in the percentage of CD4+ T lymphocytes, immune CD45+ cell infiltration into renal tissue, and changes in Na+, K+- ATPase (NKA) expression in both renal tissue and peripheral blood mononuclear cells (PBMCs). To determine whether the observed changes resulted from HS intake, high blood pressure, or both, hydralazine (HDZ) was used to lower blood pressure. The oVx HS rats received two HDZ schedules either to prevent or to treat hypertension. NKA was overexpressed in the kidneys of all oVx groups and in PBMCs of oVx HS rats. This pattern was not altered with HDZ treatment. Changes in CD4+ T lymphocytes and renal infiltration of CD45+ cells were not reversed either. High salt, but not high blood pressure, induces immune cell activation and renal infiltration. Overexpressed NKA is the primary event, and HS is the perturbation to the system in this model of SSH, which resembles the postmenopausal state.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Tumor-associated CD8+T cell tolerance induced by erythroid progenitor cells.

Introduction: CD8+T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45+ erythroid progenitor cells (CD45+EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45+EPCs mediate CD8+T cell tolerance remains incompletely understood and requires further research. Methods: In this study, the antigen-processing abilities of CD45+EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45+EPCs on CD8+T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45+EPC mediated tolerance of CD8+T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45+EPC mediated tumor-promoting effect. Results and discussion: We found that CD45+EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8+T cell anergy. In addition, we found that CD45+EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8+ T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45+EPCs during CD8+T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8+T cell tolerance in tumor-bearing mice is induced by CD45+EPCs. The results of this study have direct implications for tumor immunotherapy.

Characterization of CD34+ Cells from Patients with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) Using a t-Distributed Stochastic Neighbor Embedding (t-SNE) Protocol.

Using multi-color flow cytometry analysis, we studied the immunophenotypical differences between leukemic cells from patients with AML/MDS and hematopoietic stem and progenitor cells (HSPCs) from patients in complete remission (CR) following their successful treatment. The panel of markers included CD34, CD38, CD45RA, CD123 as representatives for a hierarchical hematopoietic stem and progenitor cell (HSPC) classification as well as programmed death ligand 1 (PD-L1). Rather than restricting the evaluation on a 2- or 3-dimensional analysis, we applied a t-distributed stochastic neighbor embedding (t-SNE) approach to obtain deeper insight and segregation between leukemic cells and normal HPSCs. For that purpose, we created a t-SNE map, which resulted in the visualization of 27 cell clusters based on their similarity concerning the composition and intensity of antigen expression. Two of these clusters were "leukemia-related" containing a great proportion of CD34+/CD38- hematopoietic stem cells (HSCs) or CD34+ cells with a strong co-expression of CD45RA/CD123, respectively. CD34+ cells within the latter cluster were also highly positive for PD-L1 reflecting their immunosuppressive capacity. Beyond this proof of principle study, the inclusion of additional markers will be helpful to refine the differentiation between normal HSPCs and leukemic cells, particularly in the context of minimal disease detection and antigen-targeted therapeutic interventions. Furthermore, we suggest a protocol for the assignment of new cell ensembles in quantitative terms, via a numerical value, the Pearson coefficient, based on a similarity comparison of the t-SNE pattern with a reference.

Citric Acid Controls the Activity of YopH Bacterial Tyrosine Phosphatase.

Purpose: Citric acid (CA) is a tricarboxylic acid with antioxidant and antimicrobial properties. Based on previous studies, the small compound with its three carboxylic groups can be considered a protein tyrosine phosphatase inhibitor. YopH, a protein tyrosine phosphatase, is an essential virulence factor in Yersinia bacteria. Materials and Methods: We performed enzymatic activity assays of YopH phosphatase after treatment with citric acid in comparison with the inhibitory compound trimesic acid, which has a similar structure. We also measured the cytotoxicity of these compounds in Jurkat T E6.1 and macrophage J774.2 cell lines. We performed molecular docking analysis of the binding of citric acid molecules to YopH phosphatase. Results: Citric acid and trimesic acid reversibly reduced the activity of YopH enzyme and decreased the viability of Jurkat and macrophage cell lines. Importantly, these two compounds showed greater inhibitory properties against bacterial YopH activity than against human CD45 phosphatase activity. Molecular docking simulations confirmed that citric acid could bind to YopH phosphatase. Conclusion: Citric acid, a known antioxidant, can be considered an inhibitor of bacterial phosphatases.

TCRαß-depleted hematopoietic stem cell transplant and third-party CD45RA+ depleted adoptive cell therapy for treatment of post-transplant parvovirus B19 aplastic crisis.

This is a case report of a 6-year-old girl with relapsed B cell acute lymphoblastic leukemia in which adoptive cell therapy was applied successfully to treat refractory human parvovirus (HPV) B19 infection. Allogenic chimeric antigen receptor (CAR) T-cell therapy (bispecific CD19/CD22) was bridged to hematopoietic stem cell transplantation (HSCT) using a haploidentical paternal donor. However, HPV B19 DNAemia progressed and transfusion-related graft versus host disease occurred. After finding a third-party related donor with a better HLA match, haploidentical HPV B19-seropositive CD45RA+ depleted cells (16.5 × 106/kg) were administered and paternal TCRαß+ depleted stem cell were retransplanted. The HPV B19 DNAemia became negative within 1 week and the reticulocyte, neutrophil, hemoglobin, and platelet counts gradually normalized. The patient remained stable during the 1-year outpatient follow-up period. Thus, our case report highlights that persistent B19 infection can lead to pancytopenia, aplastic crisis, and graft rejection and TCRαß+ depleted haplo-HSCT is an effective means of hematopoiesis recovery. CD45RO memory T-cell therapy is the key to treating and preventing the development of refractory severe HPV B19 infection.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant.

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

BACKGROUND: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. METHODS: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." RESULTS: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. CONCLUSIONS: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Immune cells at the feto-maternal interface: Comprehensive characterization and insights into term labor.

Immune cells at the feto-maternal interface play an important role in pregnancy; starting at implantation, maintenance of pregnancy, and parturition. The role of decidual immune cells in induction of labor still needs to be understood. Published reports on this topic show heterogeneity in methods of cell isolation, assay, analysis and cellular characterization making it difficult to collate available information in order to understand the contribution of immune cells at term leading to parturition. In the present study, available literature was reviewed to study the differences in immune cells between the decidua basalis and decidua parietalis, as well as between immune cells in term and preterm labor. Additionally, immune cells at the decidua parietalis were isolated from term not in labor (TNL) or term in labor (TL) samples and characterized via flow cytometry using a comprehensive, high-dimensional antibody panel. This allowed a full view of immune cell differences without combining multiple studies, which must include variation in isolation and analysis methods, for more conclusive data. The ratio of cells found in decidua parietalis in this study generally matched those reported in the literature, although we report a lower percentage of natural killer (NK) cells at term. We report that CD4 expression on CD8- NK cells decreased in term labor compared to not in labor samples, suggesting that natural killer cells may be migrating to other sites during labor. Also, we report a decrease in CD38 expression on CD8+ CD57+ T cells in labor, indicative of cytotoxic T cell senescence. Our study provides a comprehensive status of immune cells at the decidua-chorion interface at term.

A reverse translational approach reveals the protective roles of Mangifera indica in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are chronic intestinal disorders often characterized by a dysregulation of T cells, specifically T helper (Th) 1, 17 and T regulatory (Treg) repertoire. Increasing evidence demonstrates that dietary polyphenols from Mangifera indica L. extract (MIE, commonly known as mango) mitigate intestinal inflammation and splenic Th17/Treg ratio. In this study, we aimed to dissect the immunomodulatory and anti-inflammatory properties of MIE using a reverse translational approach, by initially using blood from an adult IBD inception cohort and then investigating the mechanism of action in a preclinical model of T cell-driven colitis. Of clinical relevance, MIE modulates TNF-α and IL-17 levels in LPS spiked sera from IBD patients as an ex vivo model of intestinal barrier breakdown. Preclinically, therapeutic administration of MIE significantly reduced colitis severity, pathogenic T-cell intestinal infiltrate and intestinal pro-inflammatory mediators (IL-6, IL-17A, TNF-α, IL-2, IL-22). Moreover, MIE reversed colitis-induced gut permeability and restored tight junction functionality and intestinal metabolites. Mechanistic insights revealed MIE had direct effects on blood vascular endothelial cells, blocking TNF-α/IFN-γ-induced up-regulation of COX-2 and the DP2 receptors. Collectively, we demonstrate the therapeutic potential of MIE to reverse the immunological perturbance during the onset of colitis and dampen the systemic inflammatory response, paving the way for its clinical use as nutraceutical and/or functional food.

Cellular and molecular signaling towards T cell immunological self-tolerance.

The binding of a cognate antigen to T cell receptor (TCR) complex triggers a series of intracellular events controlling T cell activation, proliferation, and differentiation. Upon TCR engagement, different negative regulatory feedback mechanisms are rapidly activated to counterbalance T cell activation, thus preventing excessive signal propagation and promoting the induction of immunological self-tolerance. Both positive and negative regulatory processes are tightly controlled to ensure the effective elimination of foreign antigens while limiting surrounding tissue damage and autoimmunity. In this context, signals deriving from co-stimulatory molecules (i.e., CD80, CD86), co-inhibitory receptors (PD-1, CTLA-4), the tyrosine phosphatase CD45 and cytokines such as IL-2 synergize with TCR-derived signals to guide T cell fate and differentiation. The balance of these mechanisms is also crucial for the generation of CD4+ Foxp3+ regulatory T cells, a cellular subset involved in the control of immunological self-tolerance. This review provides an overview of the most relevant pathways induced by TCR activation combined with those derived from co-stimulatory and co-inhibitory molecules implicated in the cell-intrinsic modulation of T cell activation. In addition to the latter, we dissected mechanisms responsible for T cell-mediated suppression of immune cell activation through regulatory T cell generation, homeostasis, and effector functions. We also discuss how imbalanced signaling derived from TCR and accessory molecules can contribute to autoimmune disease pathogenesis.

Mast cell distribution and prevalence in the murine urinary bladder.

BACKGROUND: Mast cells have been implicated in the pathology of various urinary bladder disorders. However, the distribution of mast cells throughout urinary bladder tissue remains uncertain despite mast cell prevalence being relatively well-defined. Using a mouse tissue model, this study aims to characterise the prevalence and distribution of mast cells throughout the urinary bladder. METHODS: Bladder tissues were collected from six C57BL/6J female mice. Mast cell prevalence was quantified by flow cytometry, based on the expression of the following characteristic markers: CD45, CD117 and FcɛRIα. The toluidine blue stain assessed mast cell distribution, size, and proximity to vasculature. A repeated measures one-way ANOVA was used to evaluate the density of mast cells between the discrete layers of the urinary bladder, and an ordinary one-way ANOVA was used to assess potential differences between mast cell size across the urinary bladder wall. RESULTS: It was determined that mast cells compose less than 4% of all live leukocytes in the urinary bladder. They were also found to be more prominent in the lamina propria and detrusor muscle layers, compared to the urothelium and adventitia. In addition, 20.89% of mast cells were located near vasculature, which may be an important factor in consideration of their function and potential to contribute to various bladder pathologies, such as cystitis or overactive bladder. CONCLUSION: These findings provide a baseline understanding of mast cell prevalence and distribution throughout the urinary bladder.

Discovery of Live Cell Selective Fluorescent Probes and Elucidation of Their Mechanisms: Case Study of B Cell Selective Probe CDgB.

The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).