Mononuclear Cells Of Healthy Individuals Research Articles

Landscape of Exhausted T Cells in Tuberculosis Revealed by Single-Cell Sequencing.

Tuberculosis, a contagious bacterial infection caused by Mycobacterium tuberculosis, is a substantial global health problem, impacting millions of lives annually. Exhausted T-cell signatures are critical for predicting clinical responses to tuberculosis infection. To obtain a panoramic transcriptional profile of T cells, we performed single-cell RNA-sequencing analysis of CD4+ T and CD8+ T cells isolated from peripheral blood mononuclear cells of healthy individuals and patients with tuberculosis. We identified seven subsets in CD8+ T cells and eight subsets in CD4+ T cells and elucidated the transcriptomic landscape changes and characteristics of each subset. We further investigated the cell-to-cell relationship of each subgroup of the two cell types. Different signature genes and pathways of exhausted CD4+ and CD8+ T cells were examined. We identified 12 genes with potential associations of T-cell exhaustion after tuberculosis infection. We also identified five genes as potential exhaustion marker genes. The CD8-EX3 subcluster in CD8+ T-exhausted cells was identified as an exhaustion-specific subcluster. The identified gene module further clarified the key factors influencing CD8+ T cell exhaustion. These data provide new insights into T-cell signatures in tuberculosis-exhausted populations. IMPORTANCE Identifying the changes in immune cells in response to infection can provide a better understanding of the effects of Mycobacterium tuberculosis on the host immune system. We performed single-cell RNA-sequencing analysis of CD4+ T and CD8+ T cells isolated from peripheral blood mononuclear cells of healthy individuals and patients with tuberculosis to reveal the cellular characteristics. Different signature genes and pathways of exhausted CD4+ and CD8+ T cells were examined. These will facilitate a more comprehensive understanding of the onset and underlying mechanism of T-cell exhaustion during active Mtb infection.

Hypomethylation of the promoter region drives ectopic expression of TMEM244 in Sézary cells.

Sézary syndrome (SS) is an aggressive form of cutaneous T‐cell lymphoma (CTCL) characterized by the presence of circulating malignant CD4+ T cells (Sézary cells) with many complex changes in the genome, transcriptome and epigenome. Epigenetic dysregulation seems to have an important role in the development and progression of SS as it was shown that SS cells are characterized by widespread changes in DNA methylation. In this study, we show that the transmembrane protein coding gene TMEM244 is ectopically expressed in all SS patients and SS‐derived cell lines and, to a lower extent, in mycosis fungoides and in a fraction of T‐cell lymphomas, but not in B‐cell malignancies and mononuclear cells of healthy individuals. We show that in patient samples and in the T‐cell lines TMEM244 expression is negatively correlated with the methylation level of its promoter. Furthermore, we demonstrate that TMEM244 expression can be activated in vitro by the CRISPR‐dCas9–induced specific demethylation of TMEM244 promoter region. Since both, TMEM244 expression and its promoter demethylation, are not detected in normal lymphoid cells, they can be potentially used as markers in Sézary syndrome and some other T‐cell lymphomas.

OR24 Alternative splicing of HLA-C adds another layer of polymorphism to the hyperpolymorphic HLA genes

Aim Based on DNA sequencing the Human Leukocyte Antigen (HLA) gene system is the most polymorphic one amongst the human genome. For many genes it is known that isoforms can be present due to alternative splicing. In the IPD-IMGT/HLA database, a number of structural splice variants for HLA have been described and annotated, based on DNA polymorphism in the splice site consensus sequence. But data on mRNA transcript sequences are still lacking. The lack of RNA/cDNA SBT analysis thus underestimates the actual number of HLA splice variants. In the present study, we performed cDNA sequencing to study alternative splicing of HLA-C. Methods RNA was isolated from fresh peripheral blood mononuclear cells of healthy individuals and from B cell lines using the Qiagen RNeasy kit, followed by cDNA synthesis with the Superscript III first-strand synthesis system from Thermo Fisher Scientific. Sanger Sequencing was performed after HLA-C group-specific amplification of cDNA. Twelve of the fourteen different HLA-C allele groups were studied, C * 17 and C * 18 were not available. Results Sequencing of HLA-C revealed the presence of different mRNA splice variants. One of the most intriguing splice variants involved exon 5 skipping. This mRNA variant is co-expressed together with the mature spliced transcript. The presence of this variant is depending on the allele group, implicating a role for polymorphism in this alternative splicing process. Furthermore, since exon 5 encodes the transmembrane region, the translation of this mRNA variant might result in soluble HLA-C molecules. Conclusions Some HLA-C allele groups show alternative splicing resulting in exon 5 skipping. The functional implication of this exon 5 skipping on the HLA-C expression and the influence on T and NK cell recognition is further investigated.

Generation of antigen-specific cytotoxic T lymphocytes with activated B cells

Background aimsDendritic cells are well known as the most potent antigen-presenting cells. Nonetheless, their use in immunotherapy has been limited by the time-consuming and laborious steps involved in their generation in vitro. Therefore, much attention has been placed on alternative antigen-presenting cells that are relatively more convenient to manipulate. MethodsIn this study, the efficacy of B cells as antigen-presenting cells, compared with dendritic cells, in the induction of cytotoxic T lymphocytes against cytomegalovirus-specific antigens was evaluated. B cells were isolated from the peripheral blood mononuclear cells of healthy individuals, loaded with α-galactosylceramide for activation, and nucleofected with cytomegalovirus-antigen coding plasmid DNA. Antigen-nucleofected B cells or dendritic cells were cocultured with T cells for 14 days in vitro. ResultsThe proliferation of cytotoxic T lymphocytes induced by B cells was similar to that of those induced by dendritic cells. Additionally, the immunogenicity of both sets of cytotoxic T lymphocytes was similar not only in interferon-γ enzyme-linked immunospot assays but also in cytotoxicity assays. DiscussionThese observations suggest that α-galactosylceramide-loaded B cells could be used as antigen-presenting cells as an alternative to dendritic cells. Using B cells has several benefits, including cost-effectiveness and being both less time-consuming and less labor-intensive.

Regulation of gene expression of CD4+ T lymphocyte differentiation transcription factors by galectin-3 in vitro

Now a number of CD4+ T-lymphocytes, known as Th1, Th2, Treg and Th17, is currently identified and well- studied. The methods basing on the targeted regulation of differentiation process of the Th-lymphocytes that carry out the immune response polarization attract an attention of scientists dealing with a correction of immune-mediated. In the present study, endogenous beta-galactoside-binding protein of the lectin family, galectin-3, was investigated as a regulator of T-cell homeostasis. A galectin-3 is known to be actively produced by tumor cells in malignant transformation and able to influence the processes of signal transduction, cell-cell cooperation and the implementation of programmed death. As cell differentiation processes are directly connected with the regulation of gene expression, we investigated the effect of recombinant galectin-3 on expression of mRNA of transcription.factors, which guide the differentiation of CD4+ lymphocytes. The study was performed on peripheral blood mononuclear cells of healthy individuals. The gene expression levels were evaluated by a real-time PCR. In the experiments in vitro, it has been first found the recombinant galectin-3 (0.5 mg/mL) up-regulating the expression of transcription factors Gata-3 and Rorc mRNAs and down-regulating the mRNA expression of transcription factors T-bet and FoxP3. Up to a concentration of 1 mg/mL recombinant galectin-3 stimulates Th-cells by dose-dependent manner, whereas at higher concentrations stimulating effect weakens, and inhibiting action starts prevailing. Thus, one can suppose that galectin-3 through regulation of lymphocytes differentiation promote development of allergic, autoimmune and neoplastic diseases that allows us to consider the galectin-3 as a.potential target for therapy of these diseases.

Characterization of human invariant natural killer T cells expressing FoxP3

Recently described forkhead box protein 3 (FoxP3) transcription factor is a key molecule in CD4+ CD25hi+ T-cell characterization. Invariant NK T (iNKT) cells are also characterized as regulatory cells modulating the immune response by rapidly producing T(h)1 and T(h)2 cytokines. We aimed to analyze cellular markers important in regulatory features of human iNKT cells and to study their role in functional assays. iNKT cells were single cell sorted from peripheral mononuclear cells of healthy individuals after immunostaining of invariant TCR α-chain. We found FoxP3 expression in human iNKT clones. Randomly selected iNKT cell clones (CD4+, double negative, CD8+) expressed FoxP3 mRNA and protein at different levels upon stimulation as supported by various approaches. FoxP3 mRNA and protein expression was detected in unstimulated iNKT cells as well. Furthermore, different stimulations changed the FoxP3 expression in iNKT cells over time and the most dramatic changes were observed upon anti-CD3 stimulation. Both the supernatant of iNKT cells and iNKT cells themselves exerted similar stimulation effects on PBMC proliferation in functional assays and these stimulations showed a negative correlation with FoxP3 expression. Our data indicate that the FoxP3 expression in iNKT cells may be a key transcriptional factor in controlling the regulatory function of the iNKT cells.

In Vitro Induction of HLA-G Expression In Peripheral Blood Mononuclear Cells of Healthy Individuals and Examination of Its Immunomodulatory Role

AbstractAbstract 4691HLA-G is a nonclassic MHC class I with unique features that functions as an immunomodulatory molecule. HLA-G was found to be expressed in extravillous trophoblasts during pregnancy and protects fetal tissues from the maternal immune system. In healthy adults its expression is limited on thymus and cornea. HLA-G gene is regulated with epigenetic alterations, with hypermethylation on the CpG islands of its 5′ promoter region. Our team has previously isolated and characterized naturally occurring HLA-G expressing cells (HLA-G+) with immunoregulatory properties in peripheral blood of healthy individuals which are detected on the population of monocytes. On the contrary, HLA-G is not expressed in T-lymphocytes of healthy individuals. The aim of this study was the induction of HLA-G expression with the use of demethylating agents (5-Aza-dC) in HLA-G negative (HLA-G-) peripheral blood mononuclear cells of healthy individuals and the investigation of the possible immunoregulatory properties of these induced HLA-G+ T-lymphocytes (CD3+/HLA-G+). Peripheral blood monuclear cells (PBMC) were obtained from volunteer donors and were treated with 5-Aza-dC for 3 days at final concentrations of 100nM, 500nM, 1μ M, 5μ M, 10μ M. Moreover, we investigated the potential effect of 5-Aza-dC on HLA-G expression in activated T-lymphocytes after their incubation with PHA. Then cells were analyzed by FACS for membrane HLA-G expression both in whole PBMC and CD3 cells and we analyzed their viability with the use of propidium iodide (PI) by FACS. The expression levels were compared with those of untreated cells. Moreover, the supernatant of these cultures was analyzed for the expression of the soluble form of HLA-G (sHLA-G) with Elisa, while the transcription level of HLA-G gene was tested with Reverse-Transriptase PCR by using specific primers for the membrane and soluble form of the molecule. After the induction of the expression, with the use of 5-Aza-dC, we isolated CD3+/HLA-G+ and CD3+/HLA-G- cell populations by FACS, sorting and analyzing their tolerogenic properties by using them as third party cells in mixed lymphocytes cultures (MLC). The lymphoproliferative response in the cultures was measured by using the CFSE cell proliferation assay. The incubation of PBMC with 5-Aza-dC 10μ M indicated significant increase in the percentage of membrane HLA-G expression, both in whole PBMC and in CD3+ cells, compared to the control. Specifically, the mean percentage in PBMC was 13,37% versus 5,34% of the control (p=0,0298) and in the CD3+ subpopulation was 28,21% versus 0,98% of the control (p=0,0044), with viability 20%. The incubation of activated lymphocytes with lower concentration of 5-Aza-dC (500nM) led to the increase of HLA-G expression on the CD3+ subpopulation to the level of 20,33% versus 5,31% of the control, with better viability 56,51%. The induction of HLA-G protein expression on the cells, after the incubation, was proved on molecular level with RT-PCR. Moreover, we observed an increase of sHLA-G in the supernatant of the cell cultures which contained 5-Aza-dC. Finally, starting experiments showed that the addition of CD3+/HLA-G+ cells as third party cells on MLC, suppress the proliferation when compared to the control. Our results indicate the possibility to create in vitro induced HLA-G+ T-lymphocytes with immunoregulatory properties, after treatment with the demethylating agent 5-Aza-dC. Our long term goal is the use of this population as immunosuppressive therapy. Disclosures:No relevant conflicts of interest to declare.

HHV‐6 IgG4 isotype response following measles infection

Human herpesvirus 6 (HHV-6) is widespread in the human population by infecting most individuals in early childhood. After primary infection, HHV-6 establishes a latent infection by remaining in circulating mononuclear cells of healthy individuals. The HHV-6 antibody titer increases after primary infection with measles virus. The present study was undertaken to determine the specific antiviral IgG1, IgG2, IgG3, and IgG4 subclass response patterns to HHV-6 in HHV-6-seropositive individuals with natural measles virus infection, measles vaccination, and rubella virus infection. The purpose of this study was to examine HHV-6-specific IgG isotype response in patients with acute virus coinfection. Serum samples were obtained from individuals who were seropositive for HHV-6 after natural primary infection with measles virus during an outbreak, measles vaccination, or rubella virus infection, and from healthy individuals. Sera were examined by indirect immunofluorescence assays for detection of HHV-6-specific IgG1, IgG2, IgG3, and IgG4 antibodies. A high percentage (69%) of those infected with measles virus had an HHV-6 IgG1 and IgG4 response (P < 0.001, chi(2) test), whereas persons vaccinated against measles, those infected with rubella, and healthy individuals showed an HHV-6 IgG1 response. These results demonstrate that natural measles virus infection induces an HHV-6 IgG isotype response, which suggests a shift in immune activity from a Th1 to a Th2 response. J. Med. Virol. 82:396-399, 2010. (c) 2010 Wiley-Liss, Inc.

HLA-DRB1 ⁎04 gene polymorphisms and expressions profiles of interleukin-18 and interleukin-18 binding protein following in vitro stimulation in human peripheral blood mononuclear cells of healthy individuals and patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic arthritic condition that can lead to deformities and disabilities. Interleukin-18 (IL-18) is a proinflammatory cytokine known to play a role in the acute and chronic inflammatory phases of RA. IL-18 binding protein is the natural antagonist of IL-18 protein. We aim to identify the effect of HLA-DRB1 ⁎04 gene polymorphisms on IL-18 and IL-18BP gene expressions profiles as well as the time-course profiles following in vitro stimulation with mitogens. Peripheral blood mononuclear cells from 16 RA patients and 21 healthy controls were cultured for 1, 4, 8, 12, 24, 48 and 72 h following stimulation with either LPS or PHA. mRNA expression of IL-18 and IL 18BP were determined by quantitative real-time PCR using a comparative Ct (threshold cycle) method. IL-18 levels in supernatants were measured by enzyme-linked immunosorbent assay. Basal mRNA (4.5-fold) and protein levels of IL-18 were increased and IL-18BP mRNA expression was decreased (8-fold) in RA patients when compared to controls. Similarly, increased IL-18 levels were observed in active RA patients, whereas IL-18BP expression was increased in inactive patients. There was an increase in mRNA and protein levels of IL-18 in RA patients that peaked at 4 h and 8 h respectively following LPS stimulation. A similar profile was observed for IL-18BP; however, the expression level was higher in controls than RA patients. Persistent high production of IL-18 in RA is associated with disease progression and IL-18 BP seems to inhibit this activity.

Immunomodulatory activity of Semecarpus anacardium extract in mononuclear cells of normal individuals and rheumatoid arthritis patients

Semecarpus anacardium (SA) Linn. (family Anacardiaceae), is a plant well-known for its medicinal value in Ayurveda. The nut extracts of this plant have been traditionally used as antihelminthic, anti-fungal, anti-carcinogenic and in the treatment of nervous debilities and arthritis. In this study we have evaluated crude ethanolic extract of SA nuts for its anti-inflammatory activities in vitro using peripheral blood and synovial fluid mononuclear cells of healthy individuals and rheumatoid arthritis (RA) patients . SA extract inhibited the spontaneous and LPS induced production of proinflammatory cytokines IL-1β and IL-12p40 but had no effect on TNF-α and IL-6 production, both at protein and mRNA level. The crude extract also suppressed LPS induced nuclear translocation of transcription factors, NF-κB and AP-1; the inhibition of NF-κB was through the inhibition of IκBα phosphorylation. The extract also suppressed LPS activated nitric oxide production in mouse macrophage cell line, RAW 264.7. Our results for the first time show that SA extract can inhibit proinflammatory cytokine production and demonstrate its mechanism of action.

U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture.

Human herpesvirus 6 (HHV-6) like other herpesviruses, expresses sequentially immediate early (IE), early, and late genes during lytic infection. Evidence of ability to establish latent infection has not been available, but by analogy with other herpesviruses it could be expected that IE genes that regulate and transactivate late genes would not be expressed. We report that peripheral blood mononuclear cells of healthy individuals infected with HHV-6 express the U94 gene, transcribed under IE conditions. Transcription of other IE genes (U16/17, U39, U42, U81, U89/90, U91) was not detected. To verify that U94 may play a role in the maintenance of the latent state, we derived lymphoid cell lines that stably expressed U94. HHV-6 was able to infect these cells, but viral replication was restricted. No cytopathic effect developed. Furthermore, viral transcripts were present in the first days postinfection and declined thereafter. A similar decline in the level of intracellular viral DNA also was observed. These findings are consistent with the hypothesis that the U94 gene product of HHV-6 regulates viral gene expression and enables the establishment and/or maintenance of latent infection in lymphoid cells.

Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA.

Dendritic cells (DC) generated from the peripheral blood mononuclear cells of healthy individuals or from cancer patients transfected with carcinoembryonic antigen (CEA) mRNA stimulate a potent CD8+ cytotoxic T lymphocyte (CTL) response in vitro. DCs are effectively sensitized with RNA in the absence of reagents commonly used to facilitate mammalian cell transfection. RNA encoding a chimeric CEA/LAMP-1 lysosomal targeting signal enhances the induction of CEA-specific CD4+ T cells, providing a strategy to induce T-help that may be necessary to generate and/or maintain an optimal CD8+ CTL response in vivo. CEA RNA-transfected DCs also serve as effective targets in cytotoxicity assays, thus providing a general method for inducing, as well as measuring, CEA-specific CTL responses across a broad spectrum of HLA haplotypes.

High prevalence of HHV‐6 DNA in peripheral blood mononuclear cells of healthy individuals detected by nested‐PCR

The aim of the study was the evaluation of human herpesvirus-6 (HHV-6) infection rate and semiquantification of viremia in healthy people. Healthy blood donors were studied. Human herpesvirus-6 IgG and IgM antibody titers were measured by indirect immunofluorescence assay. Human herpesvirus-6 DNA amplification (nested-PCR) was performed in peripheral blood mononuclear cells (PBMC) and in serum. Seventeen of 50 (34 percent) individuals were positive for IgG anti-HHV-6 and the titers ranged from 1:40 to 1:160. None of 30 individuals was positive for IgM anti-HHV-6, suggesting no recent infection nor reactivation. Human herpesvirus-6 DNA was detected by nested-PCR amplification in peripheral blood mononuclear cells but not in sera. When 1 microgram DNA was amplified, HHV-6 DNA was detected in 8 of 20 individual (40%), but in 18 of 20 (90%) when 5 micrograms DNA were amplified. It is concluded that HHV-6 is present in a high proportion of the healthy population but in minimal amounts, and although it can be detected in 1.4 x 10(5) PBMC, 7 x 10(5) cells are necessary to detect most cases. No reactivation was observed in healthy people.

Expression of human endogenous retroviral sequences in peripheral blood mononuclear cells of healthy individuals.

The polymerase chain reaction was used to detect expression of retroviral sequences with oligonucleotide primers derived from conserved regions of the retroviral genome. Four primer pairs derived from gag and one from pol were used in amplification of reverse-transcribed total RNA prepared from peripheral blood mononuclear cells of seven blood donors. The amplification pattern was the same from each of the seven samples. Sequencing of cloned amplification products revealed that at least three subclasses of sequences related to the human endogenous retroviruses (HERV) RTVL-H, HERV-E and HERV-K, are expressed in peripheral blood mononuclear cells of healthy individuals. This has not been previously reported.

T-cell activation in Crohn's disease: Increased levels of soluble interleukin-2 receptor in serum and in supernatants of stimulated peripheral blood mononuclear cells

Serum levels of soluble interleukin-2 receptor were determined in 29 patients with active and quiescent Crohn's disease. In addition, the ability of peripheral blood mononuclear cells of 23 of these patients to generate soluble interleukin-2 receptor following mitogenic stimulation was studied in vitro. Serum soluble interleukin-2 receptor concentrations of patients with active Crohn's disease (n = 19) were significantly elevated (757 ± 438 U/ml) compared with levels in patients with inactive disease (n = 10; 412 ± 120 U/ml) and healthy control individuals (n = 40; 375 ± 102 U/ml; p < 0.003 and p < 0.0005, respectively). Serial determinations of serum soluble interleukin-2 receptor concentration in a follow-up of 11 hospitalized patients treated for highly active disease showed a decrease from 1252 ± 494 U/ml to 527 ± 193 U/ml (p < 0.004) that corresponded to clinical improvement, as assessed by Crohn's disease activity index and a reduction of inflammatory parameters. In vitro phytohemagglutinin stimulation of peripheral blood mononuclear cells derived from patients with Crohn's disease resulted in elevated soluble interleukin-2 receptor production not only in patients with active disease (3987 ± 2439 U/ml), but also in patients with inactive disease (3297 ± 2282 U/ml), compared with the amount of soluble interleukin-2 receptor produced by mononuclear cells of healthy individuals (1523 ± 1152 U/ml; p < 0.005 and p < 0.02, respectively). In addition, cultivation of mononuclear cells without mitogen resulted in higher soluble interleukin-2 receptor production in patients with active disease than in patients with inactive disease (p < 0.02). However, patients suffering from active ulcerative colitis also had significantly increased serum levels of soluble interleukin-2 receptor (1080 ± 400 U/ml) compared with the levels in patients with chronic disease (455 ± 140 U/ml; p < 0.0025). In addition, peripheral blood mononuclear cells derived from patients with ulcerative colitis produced significantly more soluble interleukin-2 receptor upon mitogenic stimulation with phytohemagglutinin (2314 ± 936 U/ml), than cells from healthy controls (1523 ± 1152 U/ml; p < 0.05). The finding of elevated soluble interleukin-2 receptor serum levels in patients with active Crohn's disease and its increased production by mononuclear cells of patients with both active and inactive disease is a further example of an alteration of the immune system in this condition; however, this alteration can also be found in other inflammatory bowel diseases.

Listeria monocytogenes reactive T lymphocytes in healthy individuals

Peripheral blood mononuclear cells of healthy individuals were enriched for T lymphocytes and stimulated with killed organisms of Listeria sp. Different strains of Listeria sp. induced comparable T cell responses independent from their pathogenicity and virulence. Evidence is presented that killed L. monocytogenes organisms lacked mitogenic activity for human B and T lymphocytes. It is concluded that Listeria reactive T lymphocytes are commonly present in healthy individuals and hence may contribute to the rare occurrence of listeriosis in normal adults.