Characteristics and prognostic significance of T regulatory lymphocytes in lymphoid malignancies

Regulatorowe limfocyty T (Treg) stanowią niewielką subpopulację komórek CD4+ odpowiedzialną za tłumienie nadmiernej odpowiedzi immunologicznej, przyczyniając się tym samym do utrzymania homeostazy układu odpornościowego. Limfocyty Treg można wykrywać na podstawie charakterystycznego immunofenotypu CD4+CD25highFOXP3+. W wielu przypadkach nowotworów litych (np. rak jelita, płuca, jajnika) obserwowano zwiększenie liczby limfocytów Treg i stan ten niekorzystnie wpływał na rokowanie. Limfocyty Treg, poprzez swoje działanie supresorowe na limfocyty T obecne w mikrośrodowisku guza, upośledzają odpowiedź odpornościową skierowaną przeciwko komórkom nowotworowym, a więc utrudniają możliwość ich eliminacji. Sytuacja nie jest tak jasna w przypadkach, w których punktem wyjścia rozwoju nowotworu są komórki układu immunologicznego. Wyniki dotychczasowych nielicznych badań wskazują, że w złośliwych rozrostach z dojrzałych limfocytów B liczba tkankowych lub obwodowych limfocytów Treg może być podwyższona lub obniżona; podobnie ich związek z rokowaniem może być zarówno pozytywny, jak i negatywny. Nie można więc przez prostą analogię do nowotworów litych wnioskować o funkcji limfocytów Treg w chłoniakach. Na podstawie badań in vitro postuluje się wręcz, że limfocyty Treg mogą pełnić funkcję kontrolną wobec chłoniakowych limfocytów B, nie dopuszczając do klonalnego rozrostu tych ostatnich. Wskazywałoby to na zupełnie nową i jeszcze słabo poznaną funkcję limfocytów Treg. Celem pracy jest podsumowanie wyników publikacji, w których oceniano rolę Treg w nowotworach układu chłonnego, oraz omówienie kontrowersji dotyczących związku Treg z rokowaniem w tej grupie nowotworów.Regulatory T lymphocytes (Treg) represent a small subpopulation of CD4+ cells responsible for inhibition of excessive immunologic response, contributing in this way to immune homeostasis. Treg cells are characterized by CD4+CD25highFOXP3+ immunophenotype. In many cases of solid tumors (e.g. colon cancer, lung cancer, ovarian cancer) the number of Treg cells is increased and its elevation is associated with unfavorable prognosis. Treg cells by their capacity to suppress other T lymphocytes present in tumor microenvironment impair immunologic response against tumor cells thus contributing to immune evasion by malignancies. The matter is more complicated in cases where the tumor originates from immune cells. Results from published studies evaluating Treg cells in B-cell malignancies are scarce and they show that the numbers of tissue or peripheral Treg cells can be either decreased or increased. Similarly, Treg number may be associated with either positive or negative prognosis. Thus, Treg function in lymphomas should not be considered by simple analogy to solid tumors. On the basis on in vitro studies it was postulated that Treg cells can control malignant B cells, preventing clonal proliferation of the latter. Therefore it is possiblethat Treg cells may play a new, so far poorly understood role in lymphoid malignancies. The aim of this paper is to review the original articles that analyzed Treg lymphocytes in lymphoid neoplasms and to discuss the controversies on associations of Tregs with prognosis in this setting

Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer’s disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer’s disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer

Cytokines and growth factors secreted by CD105⁺CD34^- cells <i>in vitro</i>.

<p>(A) An exemplary image of a membrane used for the analysis of 80 cytokines and growth factors secreted by CD105⁺CD34^- cells. (B) Densitometric analysis of 80 cytokines and growth factors secreted by CD105⁺CD34^- cells indicates that CD105⁺CD34^- cells secrete mainly IL-6, IL-8, and GRO molecules (n = 5).</p

Changes in a size of the scar and fibrosis in the post-infarction heart of a mouse 7 weeks after MI.

<p>(A B C) Representative images illustrating a post-infarction scar in the mouse heart in a (A) Sham group or after administration of (B) PBS^- or (C) CD105⁺CD34^- cells. The images are composed of several shots (magn. 4x). (D) The area of the post-infarction scar is significantly smaller after administration of CD105⁺CD34^- cells compared to the control group, in which PBS^- was administered (n = 14); **p<0.01 by the U Mann–Whitney test. (E F G) Representative images illustrating collagen (pink) deposited between the fibers of the muscle tissue (green) in the mouse heart in a (E) Sham group or after administration of (F) PBS^-, or (G) CD105⁺CD34^- cells. The sections with the greatest ratio of post-infarction scar to the whole stained area were used for examination (magn. 40x). (H) A statistically significant reduction of fibrosis was observed in the border area of the post-infarction scar after administration of CD105⁺CD34^- cells compared to the control group after administration of PBS^- (n = 14); **p<0.01 by the U Mann–Whitney test.</p

Changes in left ventricular ejection fraction (LVEF) 6 weeks after the cells administration.

<p>LVEF increased by 5.75% between treated groups at 49 day. Day 0 –LVEF (baseline); day 7 –LVEF seven days after MI induction, the day of administration of CD105⁺CD34^- cells or PBS^-: day 49 –LVEF on the day of the collection of the hearts. PBS^-: n = 16; CD105⁺CD34^-: n = 18; **p<0.01; *p<0.05; # p = 0.0946. Comparisons between groups and within groups were performed by the one-way analysis of variance (ANOVA) with repeated measures with post-hoc Tukey tests.</p

Capillary density in the border area of the post-infarction scar 7 weeks after MI.

<p>(A B C) Representative images presenting the number of blood vessels in the border area of the post-infarction scar (magn. 20x) (A) in a Sham group, (B) in mice after administration of PBS^-, (C) in mice after administration of CD105⁺CD34^- cells. (D) In the area bordering the post-infarction scar a significant increase in the number of blood vessels in mice after administration of CD105⁺CD34^- cells compared to the control mice after administration of PBS^- was observed. n = 7; * p<0.05 by the Student’s t-test.</p

MSC-CM increased the expression of CD206 in BMDM.

<p>The incubation of MSC-CM with BMDM greatly increased the percentage of CD206⁺CD86⁺ and CD206⁺CD86^- macrophages in comparison with control BMDM cells and BMDM cells incubated in medium with LPS; n = 6 *p<0.05; **p<0.01 by one-way analysis of variance (ANOVA), using Tukeys’ multiple comparison test for post hoc analysis for the groups: CD86⁺CD206^- and CD86⁺CD206⁺; by Kruskal–Wallis one-way analysis of variance, using multiple comparison of mean ranks for all groups, for CD86^-CD206⁺ group.</p