Expression Of Functional Molecules Research Articles

Tim-3 regulates the immunosuppressive function of decidual MDSCs via the Fyn-STAT3-C/EBPβ pathway during Toxoplasma gondii infection.

Myeloid-derived suppressor cells (MDSCs) play a key role in maintaining maternal-fetal tolerance for a successful pregnancy, but the role of MDSCs in abnormal pregnancy caused by Toxoplasma gondii infection is unknown. Herein, we revealed a distinct mechanism by which T-cell immunoglobulin domain and mucin domain containing protein-3 (Tim-3), an immune checkpoint receptor that balances maternal-fetal tolerance during pregnancy, contributes to the immunosuppressive function of MDSCs during T. gondii infection. The expression of Tim-3 in decidual MDSCs was significantly downregulated following T. gondii infection. The proportion of monocytic MDSCs population, the inhibitory effect of MDSCs on T-cell proliferation, the levels of STAT3 phosphorylation, and the expression of functional molecules (Arg-1 and IL-10) in MDSCs were all decreased in T. gondii-infected pregnant Tim-3 gene knockout (Tim-3KO) mice compared with infected pregnant WT mice. After treatment with Tim-3-neutralizing Ab in vitro, the expression levels of Arg-1, IL-10, C/EBPβ, and p-STAT3 were decreased, the interaction between Fyn and Tim-3 or between Fyn and STAT3 was weakened, and the binding ability of C/EBPβ to the promoters of ARG1 and IL10 was decreased in human decidual MDSCs with T. gondii infection, while opposite results were observed following treatment with galectin-9 (a ligand for Tim-3). Inhibitors of Fyn and STAT3 also downregulated the expression of Arg-1 and IL-10 in decidual MDSCs and exacerbated adverse pregnancy outcomes caused by T. gondii infection in mice. Therefore, our studies discovered that the decrease of Tim-3 after T. gondii infection could downregulate the functional molecules of Arg-1 and IL-10 expression in decidual MDSCs through the Fyn-STAT3-C/EBPβ signaling pathway and weaken their immunosuppressive function, which eventually contribute to the development of adverse pregnancy outcomes.

Expression of Functional Molecule on Plasmacytoid Dendritic Cells Is Associated With HBsAg Loss in HBeAg-Positive Patients During PEG-IFN α-2a Treatment.

ObjectiveThe ideal endpoint of antiviral therapy in chronic hepatitis B (CHB) patients is to clear hepatitis B surface antigen (HBsAg). This study aimed to evaluate whether the expression of functional molecules on plasmacytoid dendritic cells (pDCs) is associated with HBsAg loss in HBeAg-positive patients during peginterferon alpha-2a (PEG IFN α-2a) therapy.MethodsA single-center prospective cohort study was performed in HBeAg-positive CHB patients who were treated with PEG-IFN α-2a and followed up for 4 years. HBsAg clearance, HBeAg loss and undetectable HBV DNA achieved by PEG-IFN α-2a therapy was considered as functional cure. The frequencies of pDC and CD86+ pDC in peripheral blood, and the mean fluorescence intensity of CD86 (CD86MFI) on the surface of pDC were measured at starting therapy, after 12 and 24 weeks of therapy.ResultsOf 63 patients enrolled, 17 patients achieved HBsAg loss. The baseline HBV DNA load in Non-functional-cure group was significantly higher than that in Functional cure group, and the CD86+ pDC% was significantly lower in patients without functional cure. HBV DNA load (OR=0.146, P = 0.002) and CD86+ pDC% (OR=1.183, P = 0.025) were independent factors associated with functional cure confirmed by binary logistic regression analysis. In the Functional cure group, HBsAg, HBeAg, and HBV DNA loads decreased remarkably after 12 weeks and 24 weeks of treatment compared to baseline. In Non-functional-cure group, CD86+ pDC% and CD86MFI increased significantly from baseline after 12 weeks of treatment. In the Functional cure group, compared with baseline, pDC% increased significantly at 24 weeks, while CD86MFI increased significantly after 24 weeks of treatment.ConclusionThe lower the baseline HBV DNA load and the more the baseline CD86+ pDC%, the easier it is for patients to obtain functional cure.

165P Dynamic evolution of blood immune-inflammatory descriptors in advanced non-small cell lung cancer undergoing first-line immunotherapy-based regimens

The groundbreaking results of Immune Checkpoint Inhibitors (ICIs) in NSCLC still involve a limited subset of cases, thus imposing an optimization of patient selection. With the aim to non-invasively intercept tumor-host events implicated in cancer immune surveillance and response to immunotherapy, we explored the dynamic of blood immune-inflammatory markers in a cohort of advanced NSCLC treated with first-line ICIs. Peripheral blood was prospectively collected at baseline (T0) and at first radiological disease assessment (T1) from 47 consecutive NSCLC patients undergoing first-line ICI-based therapy. We performed a flow-cytometric analysis of circulating CD3+, CD8+, CD4+, NK, NKT and Tregs as their expression of functional molecules (PD-1, Granzyme B [GnzB], Perforin [Perf]) and proliferative index (Ki67). Soluble PD-L1 (sPD-L1) was determined by immunoassay together with Lung Immune Prognostic Index (LIPI: LDH + derived Neutrophil-to-Lymphocyte Ratio). All these parameters were correlated to objective response rate (ORR) according to RECIST v1.1 criteria. From October 2020 to August 2021, 47 advanced NSCLC patients candidate to receive first-line ICI-based therapy were enrolled. Median age was 67.8 years (range 41-82), 66% were males and 81% underwent chemo-immunotherapy. ORR resulted 51%. Among baseline parameters, number of metastatic sites, bone lesions and poor LIPI negatively correlated with ORR (p<0.05), while a trend towards favorable response was apparent in tissue PD-L1high cases (ORR=67% vs 28% in PD-L1neg). A significant proliferative burst of CD8+PD-1+ lymphocytes carrying cytotoxic molecules (GnzB+, Perf+) coupled with sPD-L1 decline characterized responders. Conversely, CD8+ GnzB+/Perf+ and NK cells dramatically dropped in non-responders (χ2 test, p<0.01). Furthermore, the kinetic and extent of Treg counteraction, likely triggered by the expanding (Ki67+) pool of effector lymphocytes, appeared to be a distinctive feature of responsive patients. Our results suggest that tracking the evolution of blood immune-inflammatory profiles may provide valuable predictors of ICI efficacy in NSCLC patients.

Nrf2 activation mediates the protection of mouse Sertoli Cells damage under acute heat stress conditions

Heat stress is known to negatively impact the reproductive process of livestock, which inevitably leads to a decline in animal fertility. Nuclear factor E2-related factor 2 (Nrf2) is an inducible transcription factor, which is essential for maintaining redox signal transmission against oxidative stress. However, there is no reliable research on the response mechanism of Sertoli Cells (SCs) against heat stress and the activation of Nrf2 when SCs are exposed to heat stress. Here, we used primary mouse SCs and SCs line TM4, along with Nrf2 specific inhibitor to determine the reaction mechanism of SCs to maintain intracellular redox homeostasis and self-survival by activating Nrf2. We found that acute heat stress only affected the vitality of SCs and the expression of functional molecules (tight junction-associated proteins and lactate dehydrogenase A [LDHA]) but did not cause cell apoptosis. When Nrf2 was inhibited, more cell death occurred in TM4 cells post heat stress treatment, along with a greater decrease in cell viability and a significant increase in intracellular ROS levels. Our study clarified for the first time the protective effect of Nrf2 activation on heat stress-induced SCs damage. It explained the possible reasons or mechanisms involved in the survival of SCs, the critical protective cells in the testis, which were not affected by heat stress. This study further improved the response mechanism of SCs in the reproductive injury caused by a high-temperature environment.

Quantitation of Plasmacytoid Dendritic Cells in Chronic Hepatitis B Patients with HBeAg Positivity During PEG-IFN and Entecavir Therapy.

Plasmacytoid dendritic cells (pDCs) are crucial for control of chronic hepatitis B (CHB) virus infection. In this study, we evaluated the frequencies of pDCs and expression of functional molecules on pDCs in patients treated with PEG-IFN-α-2a or entecavir (ETV) and investigated changes during treatment. The mean fluorescence intensity of CD86 (CD86MFI) on the surface of pDCs and frequencies of pDCs and CD86+ pDCs in peripheral blood were measured. Compared with baseline, CD86+ pDC% and CD86MFI increased obviously after PEG-IFN-α-2a treatment for 12 and 24 weeks. For patients treated with ETV, only pDC% increased observably after treatment weeks 12 and 24 (P < 0.001) compared with baseline. Hepatitis B surface antigen (HBsAg) decline was significantly associated with elevated CD86+ pDC% (r = 0.348, P = 0.015) during PEG-IFN-α-2a treatment. In the HBsAg response group, CD86+ pDC% and CD86MFI (P < 0.001) increased observably after PEG-IFN-α-2a therapy, whereas only CD86MFI had a statistically significant difference after therapy compared with baseline (12 weeks versus 0 weeks, P = 0.022; 24 weeks versus 0 weeks, P = 0.015) in the HBsAg nonresponse group. CD86+ pDC% between the 2 groups had statistically significant differences at baseline (P = 0.001) and at the treatment time points of 12 and 24 weeks (P < 0.001), respectively. For patients receiving ETV therapy, pDC% increased observably, but CD86+ pDC% decreased significantly (P < 0.001) in the HBV DNA nonresponse group during early treatment with ETV. In CHB patients, HBsAg response in PEG-IFN-α-2a therapy correlated with the increase of CD86+ pDC% and HBV DNA nonresponse in ETV treatment correlated with the decrease of CD86+ pDC%.

The crosstalk between endometrial stromal cells and macrophages impairs cytotoxicity of NK cells in endometriosis by secreting IL-10 and TGF-β.

The dysfunction of NK cells in women with endometriosis (EMS) contributes to the immune escape of menstrual endometrial fragments refluxed into the peritoneal cavity. The reciprocal communications between endometrial stromal cells (ESCs) and lymphocytes facilitate the development of EMS. However, the mechanism of these communications on cytotoxicity of natural killer (NK) cells in endometriotic milieus is still largely unknown. To imitate the local immune microenvironment, the co-culture systems of ESCs from patients with EMS and monocyte-derived macrophages or of ESCs, macrophages and NK cells were constructed. The cytokine levels in the co-culture unit were evaluated by ELISA. The expression of functional molecules in NK cells was detected by flow cytometry (FCM). The NK cell behaviors in vitro were analyzed by cell counting kit-8 and cytotoxic activation assays. After incubation with ESCs and macrophages, the expression of CD16, NKG2D, perforin and IFN-γ, viability and cytotoxicity of NK cells were significantly downregulated. The secretion of interleukin (IL)-1β, IL-10 and transforming growth factor (TGF)-β in the co-culture system of ESCs and macrophages was increased. Exposure with anti-IL-10 receptor β neutralizing antibody (αhIL-10Rβ) or αTGF-β could partly reverse these effects of ESCs and macrophages on NK cells in vitro These results suggest that the interaction between macrophages and ESCs downregulates cytotoxicity of NK cells possibly by stimulating the secretion of IL-10 and TGF-β, and may further trigger the immune escape of ectopic fragments and promote the occurrence and the development of EMS.

Mesangial Viral and Psuedoviral Immunity: Possible Involvement in the Pathogenesis of Pediatric-Onset Active Lupus Nephritis

Background: Since viral infections activate type I interferon (IFN) pathways and cause subsequent release of IFN-dependent proinflammatory chemokines/cytokines, the innate immune system plays an important role in the pathogenesis of lupus nephritis (LN) as well as systemic lupus erythematosus. With respect to this issue, mesangial chemokine/cytokine expressions via innate immunity reportedly play a pivotal role in the pathogenesis of LN. Also, it has been reported that pediatric-onset active LN is found as having more severe acute inflammation than that of adult cases. Therefore, viral or “pseudoviral” infections may be attributable, at least in part, to the pathogenesis of LN, especially in pediatric-onset cases. Methods: So far, we have examined the toll-like receptor (TLR) 3 signaling cascades treated with polyinosinicpolycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, that makes “pseudoviral” infection in cultured human mesangial cells (MCs). Results: We found that the activation of mesangial TLR3 upregulated the expression of functional molecules acting as monocyte/macrophage and lymphocyte chemoattractants in MCs. Further, intense glomerular expressions of these functional molecules were observed in biopsy specimens from children with active LN. Conclusion: These observations further support the implication of viral and “pseudoviral” immunity in the pathogenesis of active LN, especially in pediatric-onset cases.

Compositional changes of B and T cell subtypes during fingolimod treatment in multiple sclerosis patients: a 12-month follow-up study.

Background and objectiveThe long term effects of fingolimod, an oral treatment for relapsing-remitting (RR) multiple sclerosis (MS), on blood circulating B and T cell subtypes in MS patients are not completely understood. This study describes for the first time the longitudinal effects of fingolimod treatment on B and T cell subtypes. Furthermore, expression of surface molecules involved in antigen presentation and costimulation during fingolimod treatment are assessed in MS patients in a 12 month follow-up study.MethodsUsing flow cytometry, B and T cell subtypes, and their expression of antigen presentation, costimulation and migration markers were measured during a 12 month follow-up in the peripheral blood of MS patients. Data of fingolimod-treated MS patients (n = 49) were compared to those from treatment-naive (n = 47) and interferon-treated (n = 27) MS patients.ResultsIn the B cell population, we observed a decrease in the proportion of non class-switched and class-switched memory B cells (p<0.001), both implicated in MS pathogenesis, while the proportion of naive B cells was increased during fingolimod treatment in the peripheral blood (PB) of MS patients (p<0.05). The remaining T cell population, in contrast, showed elevated proportions of memory conventional and regulatory T cells (p<0.01) and declined proportions of naive conventional and regulatory cells (p<0.05). These naive T cell subtypes are main drivers of MS pathogenesis. B cell expression of CD80 and CD86 and programmed death (PD) -1 expression on circulating follicular helper T cells was increased during fingolimod follow-up (p<0.05) pointing to a potentially compensatory mechanism of the remaining circulating lymphocyte subtypes that could provide additional help during normal immune responses.ConclusionsMS patients treated with fingolimod showed a change in PB lymphocyte subtype proportions and expression of functional molecules on T and B cells, suggesting an association with the therapeutic efficacy of fingolimod.

Identification of auditory neurons by retrograde labelling for patch-clamp recordings in a mixed culture of chick brainstem

We present a method to identify specific sub-populations of auditory neurons in a mixed primary cell culture of the chicken brainstem, allowing the study of individual neurons with a known identity in vitro. To label specific afferent cell types, we injected retrograde tracers (dextrans coupled to fluorescent dyes) into either the mid-line or the superior olivary nuclei (SON) of the isolated chicken brainstem in vitro. Mid-line injections resulted in stable labelling of neurons of the nucleus magnocellularis (NM), whereas injections into the SON retrogradely labelled neurons of the nucleus laminaris (NL). The fluorescent label survives the dissociation procedure and is detectable for at least 1 week in vitro. Only about 0.1% of all cells in vitro are pre-labelled. The auditory identity of the pre-labelled neurons was confirmed with calretinin immunocytochemistry and electrophysiological recordings, where the cells had typical firing patterns of auditory brainstem neurons. In the future, this method can be combined with single cell PCR to match nuclear origin, firing patterns and the expression of functional molecules in vitro.

Mesenchymal cells are required for functional development of thymic epithelial cells

Epithelial-mesenchymal interactions have essential roles in thymus organogenesis. Mesenchymal cells are known to be required for epithelial cell proliferation. However, the contribution of mesenchymal cells to thymic epithelial cell differentiation is still unclear. In the present study, we have investigated the roles of mesenchymal cells in functional development of epithelial cells in the thymus anlage in patch (ph) mutant mice, which have a primarily defect in mesenchymal cells caused by the absence of platelet-derived growth factor receptor alpha expression. In the ph/ph thymus anlage, T cell progenitors migrate normally among the epithelial cells, however, they are severely impaired to proliferate and differentiate to CD25-positive cells. Epithelial cells of the ph/ph thymus anlage show severely impaired proliferation and expression of functional molecules, such as SCF, Delta-like 4 and MHC class II, which have crucial roles in T cell development. Moreover, the cultured ph/ph thymus anlage fails to develop into a mature organ supporting full T cell development. Addition of intact thymic mesenchymal cells to organ culture induces development of the ph/ph thymus anlage. In the cultured lobes, added mesenchymal cells contribute to form not only the capsule but also the meshwork structure mingled with epithelial cells. Our present results strongly suggest the roles of mesenchymal cells in functional development of epithelial cells in thymus organogenesis. In addition, our data suggest that mesenchymal cells are required to create the thymic microenvironment and to maintain epithelial architecture and function.

Immunomodulating effect of vitamin D3 derivatives on type-1 cellular immunity

1 alpha,25-dihydroxyvitamin D3 [Calcitriol or 1,25(OH)(2)D(3)] is an important active metabolite involved in multiple functions but its calcemic effect in vivo limits its therapeutic applications. On the other hand, 22-oxa-1 alpha,25-dihydroxyvitamin D(3) (22-oxacalcitriol or 22-Oxa-1 alpha,25-D(3)), a low calcemic analog of vitamin D3 (VitD3), has been widely used as a drug for the secondary hyperparathyroidism. Here, we investigated immunomodulating effect of these two VitD3 derivatives on the differentiation of type-1 immunoregulatory cells such as dendritic cells (DC1), cytotoxic T cells (Tc1) and helper T cells (Th1). BALB/c mouse bone marrow-derived DC (BMDC1) induced by culture with Th1 condition (GM-CSF, IL-3, IL-12 and IFN-gamma) expressed higher levels of MHC Class I and Class II molecules and co-stimulatory molecules compared with BMDC0 induced by neutral condition (GM-CSF+IL-3). In addition, BMDC1 showed stronger immunostimulating activity to induce alloantigen (H-2(d))-specific cytotoxic T lymphocytes (CTL) compared with BMDC0. However, if VitD3 derivatives were added into the culture for BMDC1 induction, the expression of functional molecules and type-1 IFNs were greatly inhibited. Moreover, VitD3 derivative-treated BMDC1 lost their immunostimulating activity to induce alloantigen-specific IFN-gamma-producing Tc1. In addition, it was demonstrated that the addition of VitD3 derivatives inhibited the differentiation of IFN-gamma-producing Th1 cells from ovalbumin (OVA)-specific naive Th cells, while it rather augmented the differentiation of IL-4- or IL-10-producing Th2 cells. There was no significant difference in immunomodulating activity between 1,25(OH)(2)D(3) and 22-Oxa-1 alpha,25-D(3). Thus, VitD3 derivatives are demonstrated to inhibit the functional differentiation of DC1, Tc1 and Th1 cells, which play a critical role in type-1 cellular immune responses.

Differential STAT3, STAT5, and NF-κB activation in human hematopoietic progenitors by endogenous interleukin-15: implications in the expression of functional molecules

AbstractDifferent forms of interleukin-15 (IL-15) have been identified and shown to elicit different transduction pathways whose impact on hematopoiesis is poorly understood. We demonstrated herein that hematopoietic CD34+ cells constitutively produced endogenous secreted IL-15 (ES-IL-15) that activated different transcription factors and controlled the expression of several functional proteins, depending on the progenitor source. Thus, nuclear factor-κ B (NF-κ B) was activated in bone marrow (BM) and cord blood (CB) progenitors, whereas signal transducer and activator of transcription 3 (STAT3) and STAT5 activation was restricted to peripheral granulocyte—colony-stimulating factor (G-CSF)—mobilized and BM progenitors, respectively. ES-IL-15 acts through autocrine/paracrine loops controlled by high-affinity receptors involving IL-15 receptor α (IL-15R α). Furthermore, ES-IL-15 was found to differentially control the expression of several functional molecules important for hematopoietic differentiation. Indeed, in BM precursors, neutralizing anti—IL-15 monoclonal antibody (mAb) inhibits the expression of the γ c chain and of the chemokine stromal derived factor-1 (SDF-1) but had no effect on vascular cell adhesion molecule 1 (VCAM-1) and β 1 integrin adhesion molecule expression. Conversely, in CB progenitors, anti—IL-15 mAb inhibited VCAM-1 and β 1 integrin expression without affecting γ c chain expression and, most important, up-regulated SDF-1 expression. In conclusion, unprimed human hematopoietic CD34+ cells secrete cell-unbound IL-15, which activates through autocrine/paracrine loop distinct signaling pathways, depending on the progenitor source, thereby influencing the expression of several molecules important in the control of hematopoiesis. (Blood. 2003;102:109-117)

Expression of functional molecules by human CD3- decidual granular leucocyte clones.

Cell surface and cytoplasmic antigen expression by 35 CD3- decidual granular leucocyte (DGL) clones, derived from human endometrial tissue in the first trimester of pregnancy, has been compared with both that of fresh CD3- decidual leucocytes and that of CD3- peripheral blood natural killer (PBNK) cell clones (n = 12). The majority of DGL clones retained the antigenic phenotype of fresh cells, although CD103 (HML-1) was expressed on 50% of DGL clones but only 17% of fresh DGL. Both cytoplasmic CD3 zeta and CD3 epsilon chains were detected in > 90% of DGL clones in the absence of cell surface CD3. Cytoplasmic CD3 zeta was present in almost all fresh CD3- DGL, whereas CD3 epsilon was not. Most DGL clones did not express surface Fc gamma receptors I-III (CD64, -32 and -16, respectively) and complement receptors (CR) types 1 and 2 (CD35 and 21, respectively), but 43% expressed CR3 (CD11b/18); in contrast, all PBNK clones were CR3+. The NK cell-associated molecules Kp43 (CD94) and the p58 molecule recognized by the HP3E4 monoclonal antibody were both present on a higher proportion of CD3- PBNK (91% and 50%, respectively) than DGL clones (31% and 14%, respectively), despite expression of CD94 by > 90% of fresh CD56+ decidual leucocytes. Five of 35 CD3- DGL clones expressed cytoplasmic CD3 zeta in the absence of expression of CD2, CD16 or the p58 molecule recognized by HP3E4. These variations between CD3- DGL and PBNK cell clones in expression of functional molecules may be related to previously reported differences in major histocompatibility complex-non-restricted cytotoxic activities between these two cell types.

Expression of Functional Molecules in Non‐Hodgkin's Lymphoma

It is well known that non-Hodgkin's lymphoma (NHL) cells express various antigens which are normally involved in a variety of functions. In addition, NHL is diverse in its proliferative capacity. To investigate the relation between these factors and the clinical picture, 45 cases of NHL were studied by immunohistochemistry using snap-frozen materials obtained before therapy. Reactivities with 27 monoclonal antibodies were examined and the results were correlated with clinical findings. The expression of surface mu and ICAM-1 in B-NHLs and CD25 in T-NHLs were significantly associated with bone marrow involvement. B-NHLs without expression of CD21(B2) and T-NHLs with CD25 were seen more frequently in cases with a LDH value of over 500 units/ml. The positivity rate of Ki-67 on B-NHLs was correlated with serum LDH value, NHL histologic classification, and overall survival. These data indicate that immunophenotyping and determination of the proliferative capacity of NHL are of value not only for confirmation of the histopathologic classification of the tumor but also for assessment of clinical behavior.