13 research outputs found

    Regulatory T-cell induction by Myeloma tumour cells.

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    <p>A. The expansion of <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> natural T<sub>Reg</sub> cells, expressed as a percentage of CD4<sup>+</sup>T-cells. T<sub>Reg</sub> cells were enumerated in the PB of healthy donors, after 7 days in CM and 7 days co-cultured with mitomycin C-treated U266B cells (n = 4). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Expansion of nT<sub>Reg</sub> cells when co-culture with HMCL results from cell division, illustrated by a representative flow cytometry plot of CFSE dilution. C. The generation of <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> T-cells, expressed as a percentage of CD4<sup>+</sup>T-cells, in a co-culture assay with mitomycin C-treated U266B cells (n = 6) with varying starting populations: PB MNC, PBL CD25 depleted and CD4<sup>+</sup>CD25<sup>-</sup> T-cells. Results demonstrate that increased generation of tumour-induced regulatory T-cells (tT<sub>Reg</sub> cells) is seen with increasing purity of the seeded population. Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. D. Representative flow cytometry plots demonstrating the generation of <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> T-cells from CD4<sup>+</sup>CD25<sup>-</sup> T-cells through cell division of de novo generated <i>FoxP3</i><sup>+</sup> T-cells in a 7 day co-culture assay with mitomycin C-treated U266B cells. E. The generation of <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> T-cells, expressed as a percentage of CD4<sup>+</sup>T-cells, in a co-culture assay of CD4<sup>+</sup>CD25<sup>−</sup> T-cells (n = 10) with mitomycin C-treated MM cell lines (U266B, JJN3, JIM3 & KMS11), an erythro-leukaemia cell line (K562) and non-heamatopoietic cell lines (Mel888 & HeLa). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test (**p<0.001, *p<0.01). F. The generation of <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> T-cells, expressed as a percentage of CD4<sup>+</sup>T-cells, in a co-culture assay with fresh BM-derived myeloma plasma cells from patient samples (n = 7). Results demonstrate that increased generation of tumour-induced regulatory T-cells (tT<sub>Reg</sub> cells) is seen with primary myeloma cells. Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test.</p

    Natural and tumour-induced regulatory T-cell plasticity.

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    <p>A. The generation of IL-17 in co-culture supernatants of CD25<sup>-</sup>CD4<sup>+</sup> sorted T-cells with HMCL, compared with HMCl alone and culture medium (n = 3). Results represent all experiments, expressed as mean±SEM. B. Representative dot-plots of IL-17 producing cells generated from re-stimulation of sorted tumour-generated and naturally occurring T<sub>Reg</sub> cells after 5 days of re-stimulation. C. The proportion of IL-17 producing cells generated from re-stimulation of sorted tumour-generated and naturally occurring T<sub>Reg</sub> cells, expressing <i>FoxP3</i> after 5 days of re-stimulation, expressed as a percentage of CD4<sup>+</sup> T-cells. Results expressed as mean±SEM.</p

    <i>In vitro</i> mechanisms of tumour regulatory T-cell induction.

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    <p>A. The generation of <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> tT<sub>Reg</sub> cells from CD4<sup>+</sup>CD25<sup>-</sup> T-cells, expressed as a percentage of CD4<sup>+</sup>T-cells, in a co-culture assay with mitomycin C-treated U266 cells with and without transwell inserts (n = 7). Results represent all experiments, represented as mean±SEM and analysed using a student t-Test. B. Surface expression of HLA-DR, ICOSL (CD275) and TGFβ by human myeloma cell lines. C. Inhibition of tT<sub>Reg</sub> cells generation from CD4<sup>+</sup>CD25<sup>-</sup> T-cells by co-culture with HMCL (n = 3) through blockade of TGFβ and IL-10 using monoclonal antibodies and Latency-associated Peptide (LAP). Results represent all experiments, expressed as mean (±SEM). D. Surface expression of ICOS by T<sub>Reg</sub> cells, presented as both percentage expression of CD4<sup>+</sup>CD25<sup>+</sup><i>FoxP3</i><sup>+</sup> cells and representative dot plots. Results represent all experiments, expressed as mean ±SEM (n = 3) and analysed using a student t-test. E. Inhibition of tT<sub>Reg</sub> cells generation from CD4<sup>+</sup>CD25<sup>-</sup> T-cells by co-culture with HMCL through blockade of anti-ICOS-L (αICOS 1, 10, 100 µM) monoclonal antibody (n = 6), expressed as percentage of CD4<sup>+</sup> T-cells and percent inhibition of tT<sub>Reg</sub> cell generation. Results represent all experiments, illustrated as median with maximum and minimum values and analysed using a student t-test.</p

    Functional characteristics of tumour-induced regulatory T-cells.

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    <p>A. Suppression of anti-CD3/anti-CD28-induced autologous T-cell proliferation by tumour-generated and naturally occurring T<sub>Reg</sub> cells (n = 3), as determined by tritiated thymine incorporation. Results expressed as counts per minute (cpm)± SEM representing assays performed in triplicate. <b>Key:</b> Unstim – resting CD4<sup>+</sup>CD25<sup>-</sup> T cells, Stim – CD3/CD28 stimulated CD4<sup>+</sup>CD25<sup>-</sup> T-cells, 4∶1 etc – ratio of stimulated autologous T-cells to T<sub>Reg</sub> cells. B. The generation of IL-10 in co-cultures of CD25<sup>-</sup>CD4<sup>+</sup> sorted T-cells and HMCL, compared with HMCl alone and culture medium (n = 6, p = 0.0004). Results represent all experiments, expressed as mean±SEM and analyzed using student t-test. C. IL-10 production by tT<sub>Reg</sub> cells after 7 days of co-cultures of CD25<sup>-</sup>CD4<sup>+</sup> sorted T-cells and HMCL. Results represent all experiments, expressed as mean±SEM (n = 3) and analyzed using student t-test. D. IL-10 production by tT<sub>Reg</sub> cells after 7 days of co-cultures of CD25<sup>-</sup>CD4<sup>+</sup> sorted T-cells and HMCL. Results represent all experiments, expressed as mean±SEM (n = 3) and analyzed using student t-test. E. Representative flow cytometry plots demonstrating the generation of IFNγ<sup>+</sup><i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> T-cells from CD4<sup>+</sup>CD25<sup>-</sup> T-cells in a 7 day co-culture assay with mitomycin C-treated U266B cells. F. The proportion of IFNγ-producing <i>FoxP3</i><sup>+</sup>CD25<sup>+</sup>CD4<sup>+</sup> T-cells detectable in the peripheral blood of age-matched controls (n = 15), patients with MM (n = 15) and tTReg cells generated <i>in vitro</i> after 7 days of co-cultures of CD25<sup>-</sup>CD4<sup>+</sup> sorted T-cells and HMCL (n = 3). Histograms represent IFNγ production by cells gated on <i>FoxP3</i>/CD25/CD4 positive staing. Results expressed as mean±SEM.</p

    The use of single armed observational data to closing the gap in otherwise disconnected evidence networks: a network meta-analysis in multiple myeloma

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    Background: Network meta-analysis (NMA) allows for the estimation of comparative effectiveness of treatments that have not been studied in head-to-head trials; however, relative treatment effects for all interventions can only be derived where available evidence forms a connected network. Head-to-head evidence is limited in many disease areas, regularly resulting in disconnected evidence structures where a large number of treatments are available. This is also the case in the evidence of treatments for relapsed or refractory multiple myeloma. Methods: Randomised controlled trials (RCTs) identified in a systematic literature review form two disconnected evidence networks. Standard Bayesian NMA models are fitted to obtain estimates of relative effects within each network. Observational evidence was identified to fill the evidence gap. Single armed trials are matched to act as each other’s control group based on a distance metric derived from covariate information. Uncertainty resulting from including this evidence is incorporated by analysing the space of possible matches. Results: Twenty five randomised controlled trials form two disconnected evidence networks; 12 single armed observational studies are considered for bridging between the networks. Five matches are selected to bridge between the networks. While significant variation in the ranking is observed, daratumumab in combination with dexamethasone and either lenalidomide or bortezomib, as well as triple therapy of carfilzomib, ixazomib and elozumatab, in combination with lenalidomide and dexamethasone, show the highest effects on progression free survival, on average. Conclusions: The analysis shows how observational data can be used to fill gaps in the existing networks of RCT evidence; allowing for the indirect comparison of a large number of treatments, which could not be compared otherwise. Additional uncertainty is accounted for by scenario analyses reducing the risk of over confidence in interpretation of results
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