DP Cells Research Articles

CD4+ CD8αα+ T cells in the gastric epithelium mediate chronic inflammation induced by Helicobacter felis

CD4+ CD8αα+ double-positive intraepithelial T lymphocytes (DP T cells), a newly characterized subset of intraepithelial T cells, are reported to contribute to local immunosuppression. However, the presence of DP T cells in Helicobacter. pylori -induced gastritis and their relationship with disease prognosis has yet to be elucidated. In this study, a chronic gastritis model was established by infecting mice with Helicobacter felis. Gastric-infiltrating lymphocytes were isolated from these mice and analyzed by flow cytometry. The frequency of DP T cells in H. felis-induced gastritis mice was higher than that in uninfected mice. The gastric DP T cells were derived from lamina propria cells but were predominantly distributed in the gastric epithelial layer. These gastric DP T cells also exhibited anti-inflammatory functions, and they inhibited the maturation of dendritic cells and proliferation of CD4+ T lymphocytes in vitro. Elimination of DP T cells simultaneously resulted in severe gastritis and a reduction of H. felis load in vivo. Finally, vaccine mixed with different adjuvants was used to explore the relationship between vaccine efficacy and DP cells. Silk fibroin as the vaccine delivery system enhanced vaccine efficacy by reducing the number of DP T cells. This study demonstrated that DP T cells perform an immunosuppressive role in Helicobacter felis-induced gastritis, and consequently, DP T cells may affect disease prognosis and vaccine efficacy.

Delayed maturation of thymic epithelium in mice with specific deletion of \u03b2-catenin gene in FoxN1 positive cells

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.

Alterations in thymocyte populations under conditions of endotoxin tolerance.

Background:Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions.Methods:Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730).Results:Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ± 4.074, 72-h: 10.600 ± 3.517, 8-day: 84.770 ± 2.228), CD4+ SP cells (control: 15.770 ± 4.419, 72-h: 44.670 ± 3.089, 8-day: 6.367 ± 0.513), and CD8+ SP cells (control: 7.000 ± 1.916, 72-h: 34.030 ± 3.850, 8-day: 5.133 ± 0.647) were obviously different at different stages of ET. The polymorphism and length of TCR β chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly.Conclusion:These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.

The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFN\u03b3-independent manner

BackgroundChronic synovial inflammation is an important hallmark of inflammatory arthritis, but the cells and mechanisms involved are incompletely understood. Previously, we have shown that CCR6+ memory T-helper (memTh) cells and synovial fibroblasts (SF) activate each other in a pro-inflammatory feedforward loop, which potentially drives persistent synovial inflammation in inflammatory arthritis. However, the CCR6+ memTh cells are a heterogeneous population, containing Th17/Th22 and Th17.1 cells. Currently, it is unclear which of these subpopulations drive SF activation and how they should be targeted. In this study, we examined the individual contribution of these CCR6+ memTh subpopulations to SF activation and examined ways to regulate their function.MethodsTh17/Th22 (CXCR3−CCR4+), Th17.1 (CXCR3+CCR4−), DP (CXCR3+CCR4+), and DN (CXCR3−CCR4−) CCR6+ memTh, cells sorted from PBMC of healthy donors or treatment-naïve early rheumatoid arthritis (RA) patients, were cocultured with SF from RA patients with or without anti-IL17A, anti-IFNγ, or 1,25(OH)2D3. Cultures were analyzed by RT-PCR, ELISA, or flow cytometry.ResultsTh17/Th22, Th17.1, DP, and DN cells equally express RORC but differ in production of TBX21 and cytokines like IL-17A and IFNγ. Despite these differences, all the individual CCR6+ memTh subpopulations, both from healthy individuals and RA patients, were more potent in activating SF than the classical Th1 cells. SF activation was partially inhibited by blocking IL-17A, but not by inhibiting IFNγ or TBX21. However, active vitamin D inhibited the pathogenicity of all subpopulations leading to suppression of SF activation.ConclusionsHuman CCR6+ memTh cells contain several subpopulations that equally express RORC but differ in TBX21, IFNγ, and IL-17A expression. All individual Th17 subpopulations are more potent in activating SF than classical Th1 cells in an IFNγ-independent manner. Furthermore, our data suggest that IL-17A is not dominant in this T cell-SF activation loop but that a multiple T cell cytokine inhibitor, such as 1,25(OH)2D3, is able to suppress CCR6+ memTh subpopulation-driven SF activation.

Direct conversion of adult human fibroblasts into functional endothelial cells using defined factors

We aimed to directly convert adult human dermal fibroblasts (aHDFs) into functional endothelial cells (ECs). Lentiviral vectors encoding endothelial transcription factors (TFs) were constructed. We examined whether five TFs (FOXO1, ER71, KLF2, TAL1, and LMO2) used for the generation of mouse induced ECs (iECs) could convert the aHDFs into human iECs. Twenty-eight days after transduction with lentiviral constructs, 32.1 ± 5.1% cells expressed vascular endothelial (VE)-cadherin. Factor screening revealed that only three factors (3F: ER71, KLF2, and TAL1) were necessary to induce VE-cadherin (+) cells (49.4 ± 3.5%). However, whole transcriptome sequencing showed that VE-cadherin (+) cells were not completely reprogrammed. Mature iECs double-positive for VE-cadherin/Pecam1 (DP cells) with a cobblestone appearance were obtained at a frequency of only 5.1 ± 0.6%. Using whole transcriptome analysis, the potential factors that could block the conversion were screened. Among candidates TWIST1-knockdown enhanced efficiency of conversion. Rosiglitazone, an inhibitor of epithelial-mesenchymal transition (EMT), also improved the conversion efficiency. Moreover, a 2nd second-stage conversion process, in which VE-cadherin (+) cells were incubated for additional two weeks, further enhanced the efficiency. The final protocol for 6 weeks yielded a conversion rate of 19.6 ± 3.0% iECs, defined by DP cells depicting the nature of mature ECs in various analyses. Further analyses revealed that the genetic and epigenetic profiles of iECs resembled those of functional ECs. Collectively, aHDFs can be converted into functional ECs through the transduction of ER71, KLF2, and TAL1, combined with two EMT inhibitors (siTWIST1 and rosiglitazone), followed by 2nd stage conversion.

Circadian Rhythm Gene PER3 Negatively Regulates Stemness of Prostate Cancer Stem Cells via WNT/β-Catenin Signaling in Tumor Microenvironment.

Prostate cancer (PCa) cells are heterogeneous, containing a variety of cancer cells with phenotypical and functional discrepancies in the tumor microenvironment, where prostate cancer stem cells (PCSCs) play a vital role in PCa development. Our earlier studies have shown that ALDHhiCD44+ (DP) PCa cells and the corresponding ALDHloCD44– (DN) PCa cells manifest as PCSCs and non-PCSCs, respectively, but the underlying mechanisms regulating stemness of the PCSCs are not completely understood. To tackle this issue, we have performed RNA-Sequencing and bioinformatic analysis in DP (versus DN) cells in this study. We discovered that, PER3 (period circadian regulator 3), a circadian rhythm gene, is significantly downregulated in DP cells. Overexpression of PER3 in DP cells significantly suppressed their sphere- and colony-forming abilities as well as tumorigenicity in immunodeficient hosts. In contrast, knockdown of PER3 in DN cells dramatically promoted their colony-forming and tumor-initiating capacities. Clinically, PER3 is downregulated in human prostate cancer specimens and PER3 expression levels are highly correlated with the prognosis of the PCa patient. Mechanistically, we observed that low levels of PER3 stimulates the expression of BMAL1, leading to the phosphorylation of β-catenin and the activation of the WNT/β-catenin pathway. Together, our results indicate that PER3 negatively regulates stemness of PCSCs via WNT/β-catenin signaling in the tumor microenvironment, providing a novel strategy to treat PCa patients.

Human Fetal Scalp Dermal Papilla Enriched Genes and the Role of R-Spondin-1 in the Restoration of Hair Neogenesis in Adult Mouse Cells.

Much remains unknown about the regulatory networks which govern the dermal papilla’s (DP) ability to induce hair follicle neogenesis, a capacity which decreases greatly with age. To further define the core genes which characterize the DP cell and to identify pathways prominent in DP cells with greater hair inductive capacity, comparative transcriptome analyses of human fetal and adult dermal follicular cells were performed. 121 genes were significantly upregulated in fetal DP cells in comparison to both fetal dermal sheath cup (DSC) cells and interfollicular dermal (IFD) populations. Comparison of the set of enriched human fetal DP genes with human adult DP, newborn mouse DP, and embryonic mouse dermal condensation (DC) cells revealed differences in the expression of Wnt/β-catenin, Shh, FGF, BMP, and Notch signaling pathways. We chose R-spondin-1, a Wnt agonist, for functional verification and show that exogenous administration restores hair follicle neogenesis from adult mouse cells in skin reconstitution assays. To explore upstream regulators of fetal DP gene expression, we identified twenty-nine transcription factors which are upregulated in human fetal DP cells compared to adult DP cells. Of these, seven transcription factor binding motifs were significantly enriched in the candidate promoter regions of genes differentially expressed between fetal and adult DP cells, suggesting a potential role in the regulatory network which confers the fetal DP phenotype and a possible relationship to the induction of follicle neogenesis.

Continuous tracking of COVID-19 patients' immune status

BackgroundCOVID-19 is threating human health worldwide. We aim to investigate the dynamic changes of immune status in COVID-19 patients with clinical evolution. MethodsSixty-one COVID-19 patients (42 mild cases and 19 severe cases, 51 cases without secondary infection as non-infection group and 10 cases with secondary bacterial/fungal infection as infection group) and 52 healthy controls (HCs) were enrolled from our hospital. Leucocyte classification, lymphocyte subsets and cytokines were detected by full-automatic blood cell analyzer and flow cytometer, respectively. ResultsUpon admission, eosinophils and lymphocyte subsets decreased significantly, while neutrophils, monocytes, basophils, IL-2, IL-6, IL-10 and IFN-γ increased significantly in COVID-19 patients compared to HCs. CD3+ T and DN (CD3+CD4−CD8−) cells appeared sustained decline, leucocytes, neutrophils and IL-10 showed sustained increase in severe group compared to mild group. Compared with the non-infection group, we observed a depletion of eosinophils, CD3+ T and CD4+ T cells, but leucocytes, neutrophils, IL-6 and IL-10 on the contrary in the infection group. Besides, in severe group of COVID-19 patients, DN cells were negatively correlated with IL-10, and DP (CD3+CD4+CD8+) cells were negatively correlated with IL-6. Lymphocytes, eosinophils, CD3+ T cells, CD4+ T cells, IL-6 and IL-10 all had great diagnostic efficacy (AUC, 0.905-0.975) for COVID-19. The laboratory indicators of COVID-19 patients with improved condition also showed a recovery trend with time. ConclusionsThe immune status of COVID-19 patients is different in each stage, and dynamic monitoring of related indicators can help predict the disease and may avoid cytokine storms.

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ Tcells.

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8+ T cells. Although, during their differentiation, activated CD8+ T cells may first lose CD28, and CD28− cells may eventually express CD57 as a subsequent step, a population of CD28+CD57+(DP) CD8+ T cells can be identified in the peripheral blood. How this population is distinct from CD28−CD57−(DN) CD8+ T cells, and from the better characterized non‐activated/early‐activated CD28+CD57− and senescent‐like CD28−CD57+ CD8+ T cell subsets is currently unknown. Here, RNA expression of the four CD8+ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to “early” highly differentiated cells, with decreased TNF and IFN‐γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co‐inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8+ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8+ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8+ T cell subsets displaying defined properties.

Hair growth-promoting effect of recombinant human sonic hedgehog proteins

BackgroundHair loss, known as alopecia, causes psychological stress and influences social interactions. At present, treatment for hair loss is somewhat limited, transient, and unpredictable. Based on the critical role of sonic hedgehog (Shh) in hair follicle development, Shh itself would be a good therapeutic candidate for the prevention of hair loss as well as promotion of hair growth.MethodsTo investigate its potential use for hair treatment, we overexpressed Shh protein in E.coli by making silent mutations in 12 amino acids from N-terminus of Shh and purified it with Ni-affinity chromatography.ResultsPurified Shh proteins were able to activate Gli-responsive luciferase reporter gene in a dose-dependent manner and induce the alkaline phosphatase (AP) activity in the presence of bone morphogenic protein 4 (BMP-4). In addition, we confirmed that the treatment of recombinant Shh proteins to human dermal papilla (hDP) cells significantly increased the expressions of Shh-responsive genes as well as hair growth-related genes. Finally, we found that the administration of Shh proteins into the dorsal skin of the mouse could promote the hair density and length.ConclusionExogenous Shh proteins induced hair-related gene expression by stimulating both Shh signaling pathway and Wnt/β-catenin pathway in DP cells, and might therefore be a potential hair promoting agent.

Keratinocytes maintain compartmentalization between dermal papilla and fibroblasts in 3D heterotypic tri-cultures.

ObjectivesReproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle‐like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle‐related cells.Materials and methodsDermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three‐dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two‐dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. ResultsThe 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core‐shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell‐cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells.ConclusionsKeratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.

Distinct pattern of peripheral lymphocyte subsets in Graves’ disease with persistency of anti-TSHR autoantibodies

Background: Graves’ disease (GD) is characterized by the production of autoantibodies against the TSHR (TRAbs). With long-term treatment, serum concentrations of TRAbs decline but in some patients, despite being clinically stable, TRAbs persist for many years.Objective: To investigate whether GD patients with persistence of TRAbs constitute a subset of patients that could be identified by phenotypic analysis of circulating lymphocytes, suggesting disease heterogeneity.Materials and methods: Peripheral blood lymphocytes (including naïve, memory and effector T and B cells, Th17, regulatory T cells (Treg), recent thymic emigrants (RTEs) and double positive CD4+CD8+ (DP) cells) were analysed by flow cytometry in a cross-sectional study in 25 clinically stable GD patients, five patients at onset of GD disease and 40 healthy donors (HDs).Results: GD patients with persistence of TRAbs showed a lower percentage of Treg and lower absolute numbers of central and effector memory CD8+ T cells than HD. No differences in RTEs were found in peripheral blood from GD patients compared to HD. Stable GD patients had higher percentage of DP cells of effector phenotype than HD.Conclusions: Using extensive phenotypic analysis of lymphocyte subpopulations, it is possible to detect changes that help to identify patients with persistent TSHR antibodies and may contribute to understand why the autoimmune response is maintained.

Characterization of human dermal sheath cells reveals CD36-expressing perivascular cells associated with capillary blood vessel formation in hair follicles

Dermal sheath (DS) is located at the outermost border of hair follicles, comprising the connective tissue sheath of these follicles; DS cells are known to contribute to hair cycling and follicle neogenesis. However, the mechanisms by which DS cells contribute to hair formation are currently unclear. We investigated the global transcriptional profile of human DS cells in early passaged culture, compared with those of human dermal papilla cells (DP cells) and dermal fibroblasts. Vascular related genes were highly expressed in DS cells, and expression of the multi-ligand receptor, CD36, was significantly higher in DS cells than in DP cells. Further analyses with whole-mount imaging technique showed that dense networks of blood capillaries were formed in the DS of human anagen hair follicles, whereas regression of blood capillaries was observed in telogen and catagen hair follicles. We found that CD36-expressing cells were present in populations of DS cells, but were rarely observed in populations of DP cells and fibroblasts. Furthermore, our results indicated that CD36-expressing DS cells may participate in angiogenesis. Therefore, we concluded that CD36-expressing DS cells may modulate blood capillaries in hair follicles, in association with hair cycling.

Ablation of core fucosylation attenuates the signal transduction via T cell receptor to suppress the T cell development

Precise glycosylation plays a crucial and distinctive role in thymic T cell development. The core fucosylation is dramatically up-regulated at the transition from CD4−CD8− (DN) to CD4+CD8+ (DP) in the thymic development. Ablation of core fucosylation in T cells did reduce the size of the thymus due to a significant loss of CD4+ SP, CD8+ SP and DP thymocytes in core fucosyltransferase (Fut8) knockout (Fut8−/−) mice. T cell receptors (TCRs) are heavily core fucosylated glycoproteins. Loss of core fucosylation of TCR contributed to the reduced phosphorylation of ZAP70 (pZAP70) in Fut8−/− DP cells was observed. Compare to the Fut8+/+OT-II DP thymocytes, pZAP70 was significantly reduced in Fut8−/− OT-II DP thymocytes with OVA323–339 stimulation. Also, the pZAP70 of Fut8+/+OT-I DP thymocytes with OVA257–264 stimulation was remarkably attenuated by treatment of the fucosidase. Upon anti-CD3/CD28 Abs stimulation, the increased apoptosis was found in Fut8−/− thymocytes compared with Fut8+/+ thymocytes. Moreover, the TCRhiCD69hi (post-positive selection thymocytes) was markedly depleted in the Fut8−/− thymus without any stimulation. The expression of CD5 was significantly down-regulated on the DP cells in the Fut8−/− thymus. Our results therefore demonstrate that ablation of core fucosylation results in the abnormal T cell development due to the attenuated signaling via TCR.

Profile of Double Positive T Cells (CD45+CD3+CD4+CD8+) in HIV-Infected Population in Santos City - Brazil

Abstract Background Double Positive T Cells (CD45+CD3+CD4+CD8+) (DP) are characterized by the presence of CD4 and CD8 receptors during the development of T lymphocytes in thymus. The presence of these immature cells in peripheral blood have been shown to be present in several diseases, including in advanced stages in HIV infections. The objective of this research was to characterize the populations of Double Positive T cells in patients infected with HIV in the city of Santos, Brazil. Methodology Analysis of 12350 peripheral blood samples from HIV patients in the city of Santos between the years 2014 and 2017. The identification of the DP cells was performed by flow cytometry with CD3, CD4, CD8 and CD45 receptor markers. Results The analysis determined the prevalence of 4.7% (585 samples) of DP T cells in the population of Santos. The mean T-lymphocyte population is 1 cell. The absolute means of this population of helper and suppressor lymphocytes are 858.43 cells/μl - 31.33% and 1243.03 cells/μl - 45.39%. Samples with values greater than 5% of DP cells represented 0.11% (14 samples), which presented mean values higher than 1000 cells/μl of suppressor lymphocytes. Only half the samples had viral copies, the others were undetectable. There was a predominance of Double Negative T cells (CD45+CD3+CD4−CD8−) 95.4% (11,784 samples). Conclusions The presence of DP T cells was not superior to Double Negative T cells. No changes were found in the absolute counts of helper lymphocytes and only half had viral copies, with lymphocyte counts without compromising. The immune status of people living with HIV is important for understanding the course of the disease and for future therapeutic interventions.

Critical role of interleukin-23 in development of asthma promoted by cigarette smoke.

It has been recently reported that cigarette smoke exposure during allergen sensitization facilitates the development of allergic asthma; however, the underlying mechanisms remain elusive. We evaluated the role of interleukin (IL-23) in a cigarette smoke extract (CSE)-induced Dermatophagoides pteronyssinus (Dp)-allergic asthma mouse model. BALB/c mice were exposed to CSE during allergen sensitization period. Anti-IL-23p19 or IL-23R antibody was administered during the sensitization period. And we evaluated several immunological responses. The expression of IL-23 and IL-23 receptor (IL-23R) was examined in lung tissue. IL-23 and IL-23R expression was increased in the airway epithelium of Dp/CSE co-administered mice. CSE administration during the sensitization promoted Dp-allergic sensitization and the development of asthma phenotypes. Additionally, the proportion of innate lymphoid type 2 cells (ILC2) was also increased by CSE and Dp co-instillation. Anti-IL-23 or IL-23R antibody treatment during allergen sensitization significantly diminished phenotypes of allergic asthma and the ILC2 population. The levels of IL-33 and thymic stromal lymphopoietin (TSLP) were also significantly reduced by anti-IL-23 or IL-23R antibody treatment. IL-23 may thus play a significant role in cigarette smoke-induced allergic sensitization and asthma development. Clinically, the increase in allergen sensitization due to cigarette exposure causes onset of asthma, and IL-23 may be important in this mechanism. KEY MESSAGES: IL-23 and IL-23R expression was increased in the lung epithelium of Dp and CSE co-exposed mice during sensitization period. The population of ILC2s was increased in Dp and CSE co-exposed mice during sensitization period. Anti-IL23 or IL-23R antibody treatment with co-administration of CSE and HDM during sensitization period significantly suppresses ILC2. In vitro, IL-23 blockade in Dp and CSE-stimulated epithelial cells suppressed IL-13 expression in ILC2.

NKT Cells in Mice Originate from Cytoplasmic CD3-Positive, CD4\u2212CD8\u2212 Double-Negative Thymocytes that Express CD44 and IL-7R\u03b1

Although natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ (DP) thymocytes, the developmental origin of CD4−CD8− (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44+CD25− (DN1) cells, after that CD44+CD25+ (DN2), finally, CD44−CD25− (DN3) and CD44− CD25+ (DN4) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3+ surface CD3− and surface CD3+TCR-β− cells in DN1-NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.

Fusigen Reduces Intracellular Reactive Oxygen Species and Nitric Oxide Levels.

Oxidative stress caused by the production of excessive cellular reactive oxygen species (ROS) and high levels of nitric oxide contribute to several human pathologies. This study aimed to examine the anti-oxidant effects of fusigen, a compound produced from Aureobasidium melanogenum. Extracts of A. melanogenum were selected as a source for the isolation of fusigen. The anti-oxidant, nitric oxide suppression, as well as the free radical scavenging activities of fusigen were tested in BEAS-2B human bronchial epithelial cell line (BEAS-2B cells) and human dermal papilla cells (DP cells) using specific fluorescence dyes and flow cytometry analysis. Cell viability was determined by the MTT assay. Fusigen did not exert cytotoxicity in the human normal BEAS-2B and DP cells at concentrations up to 100 μM. Fusigen decreased basal levels of cellular ROS, as well as the levels of ROS induced by hydrogen peroxide and ferrous ion enrichment. ROS decreasing effect was confirmed in DP cells. In addition, fusigen treatment suppressed intracellular NO levels in both BEAS-2B and DP cells. The optimal process of production of purified fusigen from A. melanogenum was determined. Fusigen exhibited a low cytotoxic effect and the potential to suppress ROS and NO. These results demonstrated that fusigen may be used for the treatment or prevention of human diseases.

Thymocyte selection: From signaling to epigenetic regulation.

During thymocyte development at the double positive stage, thymocytes are subjected to a TCR quality check process termed "thymocyte selection." TCRs with proper binding capabilities to MHC molecules (with self-peptide) are able to transduce cell survival signals and allow the continuing of development to single positive T cells. It has been known that TCRs in DP cells can transduce signals with higher efficiency than peripheral mature T cells, even though they share most of the signaling components. Recent studies have revealed some thymocyte-specific signaling modulators including Themis and Tespa1. The activation of TCR signaling during positive selection results in the activation of several key transcription factors and extensive gene expression change, which has been revealed by newly developed systemic transcriptome analysis tools, and could be used for the evaluation of positive selection process. The fate determination postpositive selection is also governed on the epigenetic level including both DNA methylation and histone modifications.