Intracellular Cytokine Expression Research Articles

Cerebrospinal fluid (CSF) CD8+ T-cells that express interferon-gamma contribute to HIV associated neurocognitive disorders (HAND).

BackgroundHIV associated neurocognitive disorders (HAND) continue to affect cognition and everyday functioning despite anti-retroviral treatment (ART). Previous studies focused on mechanisms related to monocyte/macrophage mediated inflammation. However, in the ART era, there is increasing evidence for the involvement of CD8+ T-cells in CNS pathogenesis.MethodsTo investigate the relationship between T-cell responses and neurocognitive impairment (NCI), cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T-cell intracellular cytokine (IFNγ, IL-2, TNFα) and lytic marker (CD107a) expression were assessed in HIV infected subjects who underwent comprehensive neurocognitive (NC) evaluation and either initiated or changed ART.ResultsData were collected from 31 participants at 70 visits. The frequency of cytokine expressing T-cells in CSF was significantly higher than in peripheral blood for CD4+T-cells: TNFα, IL-2, IFNγ and CD8+T-cells: IL-2 and IFNγ. Analysis of T-cell activity and NCI as a function of CSF HIV RNA levels suggested a general association between NCI, high CSF CD8+ (but not CD4+T-cell) cytokine expression and CSF HIV RNA <103 copies/ml (p<0.0001). Specifically, CSF CD8+ T-cell IFNγ expression correlated with severity of NCI (r = 0.57, p = 0.004). Multivariable analyses indicated that CSF CD8+T-cell IFNγ and myeloid activation (CD163) contributed equally and independently to cognitive status and a composite variable produced the strongest correlation with NCI (r = 0.83, p = 0.0001). In contrast, CD8+ cytolytic activity (CD107a expression) was negatively correlated with NCI (p = 0.05) but was dependent on CD4 levels >400/μl and low CSF HIV RNA levels (<103 copies/ml). In our longitudinal analysis of 16 subjects, higher CSF CD8+IFNγ expression at baseline predicted NC decline at follow-up (p = 0.02). Severity of NCI at follow-up correlated with level of residual HIV RNA in CSF.ConclusionsPresence of IFNγ expressing CD8+ T-cells, absence of cytolytic CD8+ T-cells, high myeloid activation, and failure of ART to suppress HIV replication in CSF contribute to increased risk of HAND.

The impact of HIV exposure and maternal Mycobacterium tuberculosis infection on infant immune responses to bacille Calmette-Guérin vaccination.

Objective:The objective of this study is to assess the effect of maternal HIV and Mycobacterium tuberculosis (Mtb) infection on cellular responses to bacille Calmette-Guérin (BCG) immunization.Design:A mother–infant cohort study.Methods:Samples were collected from mother–infant pairs at delivery. Infants were BCG-vaccinated at 6 weeks of age and a repeat blood sample was collected from infants at 16 weeks of age. BCG-specific T-cell proliferation and intracellular cytokine expression were measured by flow cytometry. Secreted cytokines and chemokines in cell culture supernatants were analysed using a Multiplex assay.Results:One hundred and nine (47 HIV-exposed and 62 HIV-unexposed) mother–infants pairs were recruited after delivery and followed longitudinally. At birth, proportions of mycobacteria-specific proliferating T cells were not associated with either in-utero HIV exposure or maternal Mtb sensitization. However, in-utero HIV exposure affected infant-specific T-cell subsets [tumour necrosis factor-alpha (TNF-α) single positive proliferating CD4+ T cells and interferon-gamma (IFN-γ), TNF-α dual-positive CD4+ T cells]. Levels of TNF-α protein in cell culture supernatants were also significantly higher in HIV-exposed infants born to Mtb-sensitized mothers. In the presence of maternal Mtb sensitization, frequencies of maternal and newborn BCG-specific proliferating CD4+ T cells were positively correlated. Following BCG vaccination, there was no demonstrable effect of HIV exposure or maternal Mtb infection on infant BCG-specific T-cell proliferative responses or concentrations of secreted cytokines and chemokines.Conclusion:Effects of maternal HIV and Mtb infection on infant immune profiles at birth are transient only, and HIV-exposed, noninfected infants have the same potential to respond to and be protected by BCG vaccination as HIV-unexposed infants.

Vesicles from different Trypanosoma cruzi strains trigger differential innate and chronic immune responses

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.

Intracellular Expression of Neurotrophic Factors in Mononuclear Cells of Cord Blood and G-CSF-Mobilized Peripheral Blood As a Potential Source for Cellular Therapy

Purpose: To investigate a possible therapeutic mechanism for cell therapy in the field of neurological disorders using mobilized peripheral blood mononuclear cells (mPBMCs), we compared the expression of inflammatory cytokines and neurotrophic factors in PBMCs and mPBMCs from children with cerebral palsy (CP) to those from healthy adult donors and to cord blood (CB) donated from healthy newborns.Methods: We evaluated the intracellular expression of neurotrophic factors and inflammatory cytokines in PBMCs and mPBMCs from 14 CP children and 14 healthy adult volunteer donors as well as CB mononuclear cells (CB-MNCs) donated from healthy newborns. For mPBMC, granulocyte colony stimulating factor (G-CSF, 10μg/kg/day) was administered subcutaneously for five consecutive days, and apheresis was performed on the fifth day to collect mPBMC via a central venous catheter. Both PBMC collected prior to G-CSF administration and apheresed mPBMC were cryopreserved. We performed flow cytometric analysis with intracellular staining for various cytokines after thawing PBMCs and mPBMCs as well as CBs.Results: No significant differences in expression of neurotrophic factors were found between PBMC and mPBMC. However, in cells from CP children, the expression of IL-6 was significantly increased in mPBMC as compared to PBMC, and IL-3 was significantly decreased in mPBMC as compared to PBMC. In healthy adults, the expression of both IL-1β and IL-6 were significantly increased in mPBMC as compared to PBMC. The expression of BDNF in mPBMC from CP children was significantly higher than in CB or mPBMC from healthy adults. The expression of G-CSF in mPBMC from CP children was comparable to that in CB but significantly higher than in mPBMC from healthy adults. Lower expression of IL-1β, IL-3, and IL-6 and higher expression of IL-8 and IL-9 was observed from CB and mPBMCs from CP children rather than in healthy adults. Lower expression of IL-1β, and higher expression of IL-8 was observed from mPBMCs from CP children rather than in healthy adults.Conclusion: The altered expression of neurotrophic factors and anti-inflammatory cytokines in mPBMC in CP children and CB from healthy children could provide a new potential source for cellular therapy for individuals with neurologic diseases. DisclosuresLee:Korea Healthcare Technology R&D Project (A101712), Ministry for Health & Welfare, Republic of Korea: Research Funding.

The Profile of CD4+CD25+CD127low/- Regulator T Cell and the Associated Expression of Cytokines and Transcription Factors in Immune Thrombocytopenia Murine Model

BackgroundRegulator T cell (Treg cell) may be associated with immune thrombocytopenia (ITP). Our previous study has showed that the profile of CD4+CD25+CD127low/-Treg cells presented significantly decreased in ITP patients and weakened immunosuppressive activity. This cellular defect about immune modulation may play important roles in the pathogenesis of ITP. The precise reason for these abnormalities remains being elucidated. To further investigate the role of Treg cells in ITP，we detected the profiles of Treg cells and the associated mRNA expression of cytokines and Transcription factors in CD4+CD25+ Treg cells in ITP murine model .Methods1. The profiles of Treg cell in peripheral blood in ITP patients were examined with flow cytometry through intracellular cytokines analysis. Treg cells were identified as those that were CD4+CD25+CD127low/-.2. ITP was induced by daily intraperitoneal injection of anti-platelet membrane CD41 antibody（MWReg30）into BALB/c murine and the controls were daily intraperitoneal injection of IgG antibodies.3. The proportion of Treg cells in peripheral blood and spleen mononuclear cells were measured by FCM analysis. Treg cells were identified as those that were CD4+CD25+CD127low/-4. The mRNA expression of Treg cells associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+CD25+ T cells which were enriched from spleen mononuclear cells were measured by real-time PCR.5. Statistical analysis. All values were analyzed using SPSS version 18.0 software. We used the Kolmogorov-Smirnov goodness-of-fit model to assess the normality of the data. Because of the data did not show a normal distribution, the values were presented as median (range) and statistical significance was evaluated using a Wilcoxon rank-sum test. Spearman’s test was used for correlation analysis. Differences were considered significant at P <0.05.Results1. We detected the expression of intracellular cytokines. In peripheral blood of ITP patients, the median of Treg cells was 3.9 % (3.1–6.3 %), respectively, significantly lower than the controls of which that was 6.0 % (5.3–7.85 %, p <0.05).2. The percentage of CD4+CD25+CD127low/-Treg cells was significantly lower in both splenocyte and peripheral blood of ITP murine as compared with that in normal controls (p<0.05).3. ITP murine had lower mRNA expression of IL-10, TGF-β and Foxp3 in splenocyte CD4+CD25+T cells (p<0.05). The expression of Smad7 mRNA was significantly higher than the controls. No significantly difference of the STAT5 and Akt-1 mRNA expression were observed between ITP murine and controls both in splenocyte and peripheral blood.ConclusionsOur study data supported a low Treg polarization of the immune response in ITP murine model. The lower mRNA expressions of IL-10 and TGF-β indicated that the indirect immunosuppressive effect of Treg cells had impaired. The lower mRNA expressions of TGF-β and Foxp3 accelerated effects on the proliferation and differentiation of Treg cells, and the higher mRNA expression of Smad 7 inhibited the proliferation and differentiation of Treg cells. Results indicated that disorder proliferation and differentiation of Treg cells involved in the pathogenesis of ITP. DisclosuresNo relevant conflicts of interest to declare.

Abstract 16: Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells

Abstract Background: Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal associated with the growth and metastasis of tumors. In addition, VEGF has also been implicated in the suppression of immune cell function. The purpose of this study was to characterize VEGF secretion, receptor expression and effect of soluble VEGF neutralization in human lymph node cells both at rest and during activation. Methods: Human lymph nodes derived from patients with melanoma (IRB CASE 3610) containing T cells, B cells, monocyte/macrophages and dendritic cells were cultured with anti-CD3/CD28 beads and IL-2 in order to induce an inflammatory response in vitro. Culture supernatant was assayed for the presence of soluble VEGF on days 4, 7, 11 and 14 of culture. Multiparameter FACS analysis was performed to assess the cell surface expression of VEGF receptor subtypes 1 and 2 over the 14-day culture period. In addition, intracellular expression of cytokines within effector T cells was assessed at the end of the culture. Finally, a parallel culture containing a VEGF neutralizing antibody was performed to assess the effect of VEGF neutralization on effector T cell phenotype. High-throughput T cell receptor CDR3 sequencing was used to determine the molecular sequence of the variable region of the T cell receptors (ImmunoSEQ, Seattle, WA). Results: VEGF was measureable in the culture supernatant on days 4 and beyond. The proportion of cells expressing intracellular VEGF peaked at day 4 and subsequently decreased. CD11b+(monocyte/macrophage), CD11c+ (dendritic cells) and CD19+ (B) cells had the highest proportion of cells which expressed intracellular VEGF. VEGF R1 and R2 were not constitutively expressed on resting immune cells, but receptor expression was upregulated upon activation of the T cells in vitro. Day 14 T cells demonstrated intracellular IFN-γ, IL-2 and TNF-α. The addition of VEGF-blocking antibody in the culture of MDLN cells effectively neutralized the amount of soluble VEGF detected in the culture supernatant at all days. The addition of the VEGF-blocking antibody also increased the proportion of T cells expressing intracellular IFN-γ as compared T cells in the non-VEGF blocked culture and resulted in distinct molecularly defined T cell clones. Conclusion: These results confirm that soluble VEGF is produced by immune cells during activation, even in the absence of tumor. VEGF receptor expression on lymph node immune cell subsets is constitutive, but low, and then upregulated during in vitro activation of T cells, presumably due to secondary cytokine production. Neutralization of soluble VEGF resulted in qualitative differences in effector cytokine expression and T cell receptor sequences. Whether the effect on these T cells is direct via VEGF interaction with the cognate receptor or from an indirect effect on antigen-presenting cells is currently under investigation. Citation Format: Madeleine P. Strohl, Hallie Graor, Mei Zhang, Anthony Visioni, John Ammori, Isabelle Rivers-McCue, Julian Kim. Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2014-16

N-Octanoyl dopamine transiently inhibits T cell proliferation via G1 cell-cycle arrest and inhibition of redox-dependent transcription factors.

Recently, we developed a nonhemodynamic dopamine derivative, NOD, which has profound anti-inflammatory effects in vitro. As NOD also protects rats from ischemic AKI, the present study tested whether NOD is able to modulate cellular immunity for potential use as a T cell-suppressive agent. To this end, T cells were stimulated by anti-CD3/CD28 or PMA/ionomycin in the presence or absence of different concentrations of NOD. T cell proliferation, activation markers, intracellular cytokine expression, and activation of transcription factors were assessed. Whereas T cell proliferation was inhibited significantly by NOD at Day 3, proliferation was restored at Day 7 or later depending on the NOD concentration used. Inhibition of proliferation was reflected by a diminished CD25 expression and switch from naive to memory T cells. Early TCR activation events were unaffected, yet NF-κB and AP-1 were strongly inhibited by NOD. The inhibitory effect of NOD seemed to be dependent on its redox activity, as NOT, a redox-inactive NOD derivate, did not influence proliferation. NOD displayed synergistic effects with CNIs on T cell proliferation. Our data demonstrate that NOD displays T cell-suppressive activity. In keeping with its anti-inflammatory action and its beneficial effect on ischemia-induced AKI, NOD may be an interesting drug candidate to prevent CNI-related side-effects.

Immune responses following McKenzie lumbar spine exercise in individuals with acute low back pain: a preliminary study.

This study explores the immune responses following 4 weeks of McKenzie lumbar spine exercise in individuals with acute low back pain (ALBP). Fifteen patients with ALBP and 15 healthy individuals volunteered in this study. Ten ml of peripheral blood were obtained from each patient before and after exercise sessions, and from healthy individuals at the beginning of the study. Flow cytometric analysis was used to evaluate the frequencies of CD4+ T lymphocyte sub-populations and the intracellular cytokine expression within this cell population. Pain perceptions were obtained at baseline and following each week of exercise sessions. In comparison with healthy subjects there was an elevated frequency of memory (CD4+CD45RO+) T cells, helper inducer (CD4+CD29+) T cells, CD3+CD16+CD56+ T cells and a lower frequency of naïve/suppressor (CD4+CD45RA+) T cells at base line in back pain patients (p<0.05). After 4 weeks of McKenzie exercise sessions, pain intensity significantly decreased (p<0.05); however, there was no significant difference in the frequency of memory (CD4+CD45RO+) T cells, helper inducer (CD4+CD29+) T cells, CD3+CD16+CD56+ T cells and naïve/ suppressor (CD4+CD45RA+) T cells at base line relative to these cell populations after exercise sessions. The percentage of Pan (CD3+) T cells expressing IL-8 and TNF-α and the CD3+ T cells expressing the anti-inflammatory cytokine IL-4 increased significantly (p<0.05) following exercise sessions in comparison with baseline and healthy references. The reduction in pain scores did not correlate with elevated anti-inflammatory cytokines. McKenzie exercise sessions induced an immune activation state and simultaneously up regulated anti-inflammatory IL-4 cytokines that boost pain relief.

Induction of dendritic cell maturation in the skin microenvironment by soluble factors derived from colon carcinoma

Autologous tumor cell-based vaccines provide a wide range of tumor antigens and personalized neo-epitopes based on individual tumors’ unique antigenic mutanome signatures. However, tumor-derived factors may hamper in situ maturation of dendritic cells (DC) and thus interfere with the generation of effective anti-tumor immunity. As the skin is a preferred site for tumor vaccine delivery, we investigated the influence of primary colon carcinoma-derived soluble factors on the maturation state of migrating DC in a human skin explant model. Primary tumor-derived supernatants (TDSN) enhanced the phenotypic maturation state of skin-emigrated DC, resulting in an increased T-cell stimulatory ability in an allogeneic mixed leukocyte response. In case of monocyte-derived DC a similar TDSN-induced maturation induction was found to entirely depend on cyclooxygenase (COX)-regulated prostaglandins. In contrast, the increase in skin-emigrated DC maturation was completely prostaglandin-independent, as evidenced by the inability of the COX inhibitor indomethacin to abrogate this TDSN-induced effect. Although TDSN conditioning affected a drop in IL-12p70 release by the skin-emigrated DC and induced a predominant Th17/Th22 transcriptional profile in subsequently stimulated T-cells, Th cell subset differentiation, as assessed by intracellular cytokine expression upon polyclonal priming and re-stimulation, was not affected. Comparative analysis of phenotypic and transcriptional profiles suggests that the observed maturational effects in skin-derived DC may have been induced by tumor-derived GM-CSF. In conclusion, soluble factors derived from whole-cell colon tumor vaccines will not negatively impact DC migration and maturation in human skin, but rather induce DC maturation that will facilitate the priming of a poly-functional Th cell response.

Effect of aspirin desensitization on T-cell cytokines and plasma lipoxins in aspirin-exacerbated respiratory disease

The pathogenesis of aspirin-exacerbated respiratory disease (AERD) is thought to be based on, mainly, overproduction of eicosanoid lipid mediators and on defective anti-inflammatory regulators. Aspirin desensitization treatment, the mainstay of controlling asthma and rhinitis in AERD patients, however, is the least understood aspect of the disease. The study was designed to determine the effect of aspirin desensitization on T-lymphocyte cytokine expression and on plasma lipoxin levels in AERD. Spirometry, skin-prick test and asthma control test were documented and intracellular cytokine expression in T lymphocytes and plasma lipoxin levels were measured in 23 AERD patients, 17 aspirin-tolerant asthmatic (ATA) patients, and 16 healthy controls. In the AERD group nasal symptom and smell scores were assessed. Of the 23 AERD patients 15 accepted to undergo aspirin desensitization protocol and 14 of them were desensitized successfully. In the desensitized AERD group, cytokine and lipoxin measurements were repeated after 1-month aspirin treatment. CD4(+) IL-10 levels were higher in AERD patients than in healthy controls and CD4(+) interferon (IFN) gamma levels were higher in AERD and ATA patients than in controls. Plasma lipoxin-A4 and 15-epi-lipoxin-A4 levels were similar among the three study groups. In the AERD group, subjects underwent aspirin desensitization followed by a 1-month aspirin treatment. Clinical parameters improved and CD4(+) IFN-gamma levels decreased significantly. No significant change in lipoxin levels was recorded. CD4(+) IFN-gamma and CD4(+) IL-10 levels in AERD patients after 1-month aspirin desensitization treatment were similar to the healthy controls. The study confirms aspirin desensitization is effective clinically in AERD patients and suggests that IFN gamma and IL-10 expression in CD4(+) T lymphocytes may be related to the mechanism of action.

A161: Novel 3‐Dimensional Explant Method Facilitates the Study of Lymphocyte Populations in the Synovium and Reveals a Large Population of Resident Memory T cells in Rheumatoid Arthritis

Background/Purpose:Traditionally, immunologic memory was thought to be maintained by populations of central (TCM) and effector (TEM) memory lymphocytes that circulate in the blood and lymphatics, only temporarily extravagating into peripheral tissue to execute immunologic responses. Recently, this theory of adaptive memory has been challenged by the discovery of long‐lived and stable populations of tissue‐resident memory T cells (TRM). While TRM have been implicated in the pathogenesis of skin, intestinal, and lung inflammatory diseases, little information is available about TRM in synovium. The study of TRM in arthritis has been impaired by the scarcity of synovium samples coupled with the meager yield of lymphocytes from this tissue using conventional tissue digestion protocols. We have employed novel culturing techniques, previously used to examine TRM in skin, to further characterize TRM in inflammatory arthritis.Methods:Discard synovium samples were collected from patients with rheumatoid arthritis (RA) who were undergoing joint replacement surgery. A 3‐dimensional explant method was used to isolated synovial T cells. Synovial tissue was cut into pieces with scissors, loaded on titanium matrices, and cultured in 24 well plates with T cell media enriched with IL‐2, 15. After 3 weeks, T cells were harvested. Cell surface markers were studied with flow cytometry. Intracellular cytokine expression was assessed by flow cytometry after T cells were stimulated, fixed, and permeabilized, while Foxp3 expression was evaluated following fixation and permeabilization.Results:Seven RA synovial samples were obtained from 5 women and 2 men. All patients were receiving immunomodulatory medications (prednisone n = 5, methotrexate n = 2, tumor necrosis factor inhibitors n = 1). Synovial samples were small, ranging from 0.5 mm2 to 2 mm2; yet, a substantial number of mononuclear cells were harvested (mean: 7.5 × 106). The isolated lymphocytes were phenotypically similar to prior published reports of synovial T cell populations. The majority of CD3+ lymphocytes were CD4+ (Mean ± SEM: 73.5 ± 7.1%) compared to CD8+ cells (25.5 ± 7.3%). In the CD4+ subset, interferon gamma expression was common (39.5 ± 9.0%), IL‐17+ expression was rare (range: 0.1 to 3.4%), and T regulatory cells (CD4+CD25+Foxp3+) were present (8.9 ± 3.2%). A memory phenotype characterized by CD45RO+ expression predominated in both CD4+ (94.5 ± 2.4%) and CD8+ (78.0 ± 6.8%) lymphocytes. A TRM population that lacked the TCM cell surface markers CCR7+ and CD62L+ was identified in CD4+ (47.5 + 10.6%) and CD8+ (36.8 ± 8.1%) cells.Conclusion:The 3‐dimensional explant culturing method is a novel tool, which can be employed to study lymphocytes that are resident in synovium. We confirmed that the T cells isolated through this technique were phenotypically similar to populations of RA synovial lymphocytes previously assessed by immunohistochemistry and tissue digestion. A population of TRM was identified in all synovial samples, suggesting these T cells may be important in the pathogenesis of inflammatory arthritis, though further study of this population is needed.

Brief Report: Autologous Stem Cell Transplantation Restores Immune Tolerance in Experimental Arthritis by Renewal and Modulation of the Teff Cell Compartment

Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production.

Helicobacter pylori-driven modulation of NK cell expansion, intracellular cytokine expression and cytotoxic activity

During Helicobacter pylori (Hp) infections, innate immune cells may be positively or negatively modulated by Hp compounds or by Hp-induced cytokines. We have shown previously that the natural cytotoxic activity of PBMC was lower in Hp-infected [Hp(+)] than Hp-uninfected individuals [Hp(-)]. Here, we asked whether the Hp-modulated cytotoxic amplitude is associated with changes in the number of NK cells, their activation or intracellular cytokine expression. Flow cytometry immunophenotyping of PBMC was performed with regard to the surface receptors CD3, CD56 and CD25, and intracellular cytokine expression of IL-2, IFN-γ and IL-10 after in vitro stimulation with Hp glycine acid extract (GE), Hp LPS or standard Escherichia coli LPS. Hp GE-driven enhancement of lymphocyte cytotoxic activity was associated with the expansion of CD3(-)CD56(+)CD25(+) NK cells and the up-regulation of IFN-γ and/or IL-2 synthesis, up to the higher level in Hp(-) than in Hp(+), while Hp LPS-mediated decrease in lymphocyte cytotoxicity was accompanied by the lack of CD3(-)CD56(+)CD25(+) NK propagation, the inhibition of pro-inflammatory cytokine expression and intense expansion of IL-10-producing NK cells. Thus, the cytotoxic and cytokine activities of NK cells were dependent on the type of antigenic challenge and the Hp status, that is, NK cells could be modulated positively by Hp GE Ags and negatively by Hp LPS.

Flow cytometric detection of p38 MAPK phosphorylation and intracellular cytokine expression in peripheral blood subpopulations from patients with autoimmune rheumatic diseases.

Flow cytometric analysis of p38 mitogen-activated protein kinase (p38 MAPK) signaling cascade is optimally achieved by methanol permeabilization protocols. Such protocols suffer from the difficulties to accurately detect intracellular cytokines and surface epitopes of infrequent cell subpopulations, which are removed by methanol. To overcome these limitations, we have modified methanol-based phosphoflow protocols using several commercially available antibody clones suitable for surface antigens, intracellular cytokines, and p38 MAPK. These included markers of B cells (CD19, CD20, and CD22), T cells (CD3, CD4, and CD8), NK (CD56 and CD7), and dendritic cells (CD11c). We have also tested surface markers of costimulatory molecules, such as CD27. We have successfully determined simultaneous expression of IFN-γ, as well as IL-10, and phosphorylated p38 in cell subsets. The optimized phosphoflow protocol has also been successfully applied in peripheral blood mononuclear cells or purified cell subpopulations from patients with various autoimmune diseases. In conclusion, our refined phosphoflow cytometric approach allows simultaneous detection of p38 MAPK activity and intracellular cytokine expression and could be used as an important tool to study signaling cascades in autoimmunity.

Effects of 3D nanocomposite bioceramic scaffolds on the immune response.

The interaction of new nanocomposite mesoporous glass/hydroxyapatite (MGHA) scaffolds with immune cells involved in both innate and acquired immunity has been studied in vitro as an essential aspect of their biocompatibility assessment. Since the immune response can be affected by the degradation products of bioresorbable scaffolds and scaffold surface changes, both processes have been evaluated. No alterations in proliferation and viability of RAW-264.7 macrophage-like cells were detected after culture on MGHA scaffolds which did not induce cell apoptosis. However, a slight cell size decrease and an intracellular calcium content increase were observed after contact of this cell line with MGHA scaffolds or their extracts. Although no changes in the percentages of RAW cells with low and high contents of reactive oxygen species (ROS) are observed by the treatment with 7 day extracts, this study has revealed modifications of these percentages after direct contact with scaffolds and by the treatment with 24 h extracts, related to the high reactivity/bioactivity of this MGHA nanocomposite at initial times. Furthermore, when normal fresh murine spleen cells were used as an experimental model closer to physiological conditions, no significant alterations in the activation of different immune cell subpopulations were detected in the presence of 24 h MGHA extract. MGHA scaffolds did not affect either the spontaneous apoptosis or intracellular cytokine expression (IL-2, IL-10, IFN-γ, and TNF-α) after 24 h treatment. The results obtained in the present study with murine immune cell subpopulations (macrophages, lymphocytes B, lymphocytes T and natural killer cells) support the biocompatibility of the MGHA material and suggest an adequate host tissue response to their scaffolds upon their implantation.

The Th17/Treg imbalance in patients with cardiogenic shock

Type 17 helper T (Th17) cells producing the proinflammatory signature cytokine interleukin (IL)-17 are conterregulated by regulatory T cells (Treg) producing anti-inflammatory cytokines like transforming growth factor (TGF)-β and interleukin-(IL)-10. An imbalance of the Th17/Treg-ratio toward Th17 cell subset was shown to be involved in plaque destabilization and acute myocardial infarction (AMI), while no data exist in infarction-related cardiogenic shock (CS). The objective of this study was to evaluate the role of Th17/Treg and their related cytokines in uncomplicated AMI and infarction-related CS. In an observational monocentric study, blood sample from age-matched healthy controls (HC, n=20), patients with uncomplicated AMI (n=20), patients with CS who survived for at least 28days (CS-survivors, n=20) and CS-non-survivors (n=20) were analyzed. Circulating Th17 and Treg cell subsets and their intracellular cytokine expression were measured by flow cytometry and associated with circulating proinflammatory Th17-derived cytokines IL-6, IL-17 and their anti-inflammatory Treg-derived cytokines TGF-β and IL-10 measured by enzyme immunoassay. According to the severity of ACS, CS-non-survivors showed the highest levels of Th17 (p<0.001) and the lowest levels of Treg cells (p<0.001) favoring a Th17/Treg imbalance toward the proinflammatory Th17 response (p<0.001). Changes of T cell subsets were also associated with a proinflammatory cytokine expression measured by increased IL-6 (p<0.001) and IL-17 levels (p<0.001) and decreased TGF-β (p<0.001) and IL-10 levels (p=0.057). For the Th17/Treg-ratio at admission, a cut-off point of >0.33 had a sensitivity of 90% and a specificity of 80% to determine 28-day mortality in CS (confirmed by ROC analysis, area under the curve: 0.88±0.06, p<0.001). Th17/Treg-ratio >0.33 was observed to be an independent predictor for 1-year mortality in CS confirmed by Cox proportional hazard analysis (hazard ratio (HR): 4.31; 95% confidence interval (CI) 1.44-12.93; p=0.009). The Th17/Treg imbalance toward a Th17 shift might represent a promising candidate as therapeutic target and risk indicator in cardiogenic shock.

Intracellular cytokine expression in T cells from patients with chronic lymphocytic leukemia

Functional disorders of T lymphocytes play an essential role in abnormal immune response in patients with chronic lymphocytic leukemia. The aim of this study was to assess the profile of cytokines expressed by T cells derived from patients with CLL. We have demonstrated that the intracellular levels of IL-2, IL-4, IFN-γ, TNF, IL-6 and IL-10 were significantly higher in T cells of CLL patients than in healthy donors. Moreover, the percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+, and CD4+/CD3+/IL-2+ cells were significantly higher in ZAP-70-positive patients compared with ZAP-70-negative ones. Likewise, significantly higher percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+ cells were observed in CD38-positive than in CD38-negative cases. What is more, there was a significant difference in median percentage of CD3+/CD4+ cells expressing TNF, IL-4, IFN-γ, IL-2 or IL-6 between patients carrying the 11q22.3 deletion and/or the 17p13.1 deletion and patients without these genetic aberrations. Our results confirm the functional disorders of T cells in CLL and their influence on the clinical course of the disease.

Mammaglobin B (SCGB2A1) is a novel tumour antigen highly differentially expressed in all major histological types of ovarian cancer: implications for ovarian cancer immunotherapy

Background:We studied the genetic fingerprints of ovarian cancer and validated the potential of Mammaglobin b (SCGB2A1), one of the top differentially expressed genes found in our analysis, as a novel ovarian tumour rejection antigen.Methods:We profiled 70 ovarian carcinomas including 24 serous (OSPC), 15 clear-cell (CC), 24 endometrioid (EAC) and 7 poorly differentiated tumours, and 14 normal human ovarian surface epithelial (HOSE) control cell lines using the Human HG-U133 Plus 2.0 chip (Affymetrix). Quantitative real-time PCR and immunohistochemistry staining techniques were used to validate microarray data at RNA and protein levels for SCGB2A1. Full-length human-recombinant SCGB2A1 was used to pulse monocyte-derived dendritic cells (DCs) to stimulate autologous SCGB2A1-specific cytotoxic T-lymphocyte (CTL) responses against chemo-naive and chemo-resistant autologous ovarian tumours.Results:Gene expression profiling identified SCGB2A1 as a top differentially expressed gene in all histological ovarian cancer types tested. The CD8+ CTL populations generated against SCGB2A1 were able to consistently induce lysis of autologous primary (chemo-naive) and metastatic/recurrent (chemo-resistant) target tumour cells expressing SCGB2A1, whereas autologous HLA-identical noncancerous cells were not lysed. Cytotoxicity against autologous tumour cells was significantly inhibited by anti-HLA-class I (W6/32) monoclonal antibody. Intracellular cytokine expression measured by flow cytometry showed a striking type 1 cytokine profile (i.e., high IFN-γ secretion) in SCGB2A1-specific CTLs.Conclusion:SCGB2A1 is a top differentially expressed gene in all major histological types of ovarian cancers and may represent a novel and attractive target for the immunotherapy of patients harbouring recurrent disease resistant to chemotherapy.

SAT0072 Frequency of interferon-gamma-positive TH17 (TH17-1) cells is up-regulated in synovial fluids in patients with rheumatoid arthritis

BackgroundTh17 cells are a recently identified CD4+ CD45RO+helper T cell subset that produce IL-17 with proinflammatory actions and are thought to play a critical role in the pathogenesis of collagen-induced...

AB0131 Lupus monocyte-derived dendritic cells treated with 1a,25-dihydroxyvitamin d3 and dexamethasone are tolerogenic and induce il-10 producing regulatory t cells

BackgroundThe pathogenesis of systemic lupus erythematosus (SLE) is characterized by dysregulated dendritic cells (DCs) with over-production of type I interferon and hyperactive adaptive immune response. Clinical benefits of tolerogenic DCs...