Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation

Assessment of expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LH/hCGR) and hCG protein in ovarian cancer tissues

Abstract Objectives: Ovarian cancers still remain a significant worldwide epidemiological problem. The vast majority of newly diagnosed cases are in advanced clinical stages, and the five -year survival rate in all clinical stages amounts up to less than fifty percent. A better understanding of the biology of ovarian cancer can bring us closer to successful treatment and recovery. The aim of this study was to evaluate the expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LH/hCGR) and the expression of hCG protein in the ovarian cancer tissue. Materials: Histologically proven primary ovarian cancer samples (n=20) were obtained from women undergoing gynecologic surgery. Frozen sections of ovarian cancer tissues were evaluated by means of indirect immunofluorescence. Results: The expression of hCG protein was ubiquitous throughout all samples, while LH/hCGR was detected in only sixty percent of samples. Co-expression of LH/hCGR and hCG protein was detected in some individual cells in tumor tissue. Some cancer cells expressed only hCG protein, but not LH/hCGR. Conclusions: Expression of LH/hCG receptor is a characteristic feature of various histological types of ovarian cancer. Co-expression of LH/hCGR and hCG protein in individual cancer cells may point to an autocrine or paracrine mechanism of _hCG activity in the tumor microenvironment. Further studies are needed to evaluate LH/hCGR and hCG protein function in the course of neoplastic development

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4+ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8+ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4+CD25hiFOXP3+ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8+ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8+ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8+ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8+ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection

Expression of survivin, SDF-1 and CXCR4 on tumor cells in ovarian cancer

Summary Background: epithelial ovarian cancer (EOC) has the highest mortality rate among patients with gynecologic malignancies. Lack of specific and early symptoms and of screening tests causes that most patients are diagnosed in advanced stage of disease. Radical surgery followed by chemotherapy does not bring satisfactory curative effects. Objectives: the urgent need exists to define the optimum biomarker for ovarian cancer to predict patients’ response to curative therapy. Our current study aimed at correlation between the expression of survivin, SDF-1, CXCR-4 on tumor tissue and clinical outcome of patients with ovarian cancer. Results: we showed that survivin expression correlates with histological grading of the tumor. No correlation was found in terms of SDF-1/CXCR-4 expression and clinicopathologic data. Conclusions: further studies covering larger number of patients are needed to determine whether SDF-1 and CXCR-4 might be considered as biomarkers for ovarian cancer

Tumor-Derived Microvesicles Induce, Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background: Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4 + CD25 high FOXP3 + Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/ expansion/and activation of human Treg. Methodology/Principal Findings: TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4 + CD25 neg T cells into CD4 + CD25 high FOXP3 + Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-b1, CTLA-4, granzyme B and perforin expression (p,0.05) and mediated stronger suppression of responder cell (RC) proliferation (p,0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-b1 and/or IL-10 significantly inhibited TMV ability to expand Treg. Conclusions/Significance: This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV wit

Problemy diagnostyczne u pacjentki z wodobrzuszem, podwyższonym poziomem Ca 125, przeciwciałami przeciwjądrowymi i przeciwciałami przeciw mięśniom gładkim przypominającym raka jajnika – przypadek kliniczny

Abstract The paper describes a case of 26-year-old patient primarily suspected to suffer from the ovarian or peritoneum cancer due to high level of Ca 125 antigen and ascites. During exploratory laparotomy, neoplastic process was excluded, which was confirmed by histopathological examination. Further diagnostic tests were performed. The patient was not infected with hepatitic B or C virus, and there was no biochemical evidence of liver disease. Detailed, wide biochemical and immunological investigations detected antinuclear and anti smooth muscle autoantibodies in the blood serum. Afterwards, the patient was admitted to the Department of Gastroenterology and autoimmune chronic hepatitis was confirmed.Streszczenie Artykuł ten opisuje przypadek 26-letniej pacjentki, u której pierwotnie podejrzewano rozwijającego się raka jajnika lub pierwotnego raka otrzewnej, na co wskazywał wysoki poziom Ca 125. W trakcie laparotomii zwiadowczej wykluczono proces nowotworowy, co zostało potwierdzone badaniem histopatologicznym. Następnie rozszerzono badania diagnostyczne, mające wyjaśnić przyczynę wysokiego poziomu Ca 125. Wykluczono zakażenie wirusem zapalenia wątroby typu B lub C. Nie było żadnych biochemicznych wykładników choroby wątroby. Za pomocą szczegółowych testów biochemicznych i immunologicznych wykryte zostały autoprzeciwciała przeciwjadowe i przeciwko mięśniom gładkim. Ostatecznie pacjentka została przyjęta do Kliniki Gastroenterologii i zostało rozpoznane chroniczne zapalenie wątroby o podłożu autoimmunologicznym

TMV Convert CD25^neg T cells to Treg.

<p>(<b><u>A</u></b>) Flow cytometry histograms of cultured (d5) CD4⁺CD25^neg T cells showing conversion of CD25^neg T cells into CD25⁺ T cells ± TMV or DC-derived MV (<i>left panel</i>) and expression of FOXP3 in the converted CD4⁺CD25⁺ T cells (<i>right panel</i>) in the same cultures. (<b><u>B</u></b>) A phenotypic profile of CD4⁺CD25^high T cells present in 7 day cultures of CD4⁺CD25⁺ T cells ± TMV or DC-derived MV. T cells were stained with various mAbs and evaluated by multiparameter flow cytometry. The gate is set on CD4⁺CD25^high T cells. The data are mean percentages ± SD of positive cells from three independent experiments. (<b><u>C</u></b>) MFI for FasL, IL-10, TGF-β1, granzyme B and perforin expression in CD4⁺CD25^high T cells cultured as described in (<b><u>B</u></b>) ± TMV. The data are representative of three independent experiments.</p

TMV increase suppressor activity of Treg.

<p>The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4⁺CD25^neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (<b><u>A</u></b>) and greater inhibition of RC proliferation (<b><u>B</u></b>). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD⁺ RC was lower (<b><u>C</u></b>) as was RC proliferation inhibition (<b><u>D</u></b>). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (<b><u>C</u></b>) and inhibited RC proliferation (<b><u>D</u></b>).</p