A preliminary study into the genetic transformation of carrot (daucus carota) using agrobacterium derived vectors

Agrobacterium tumefaciens infection has been used to transform different species of dicotyledonous plants. In this study experiments were carried out leading to a transformation protocol for the carrot (daucus carota). Resistance to the antibiotic kanamycin was transferred into suspension culture cells and root discs by cocultivation with Agrobacterium harbouring a binary vector system

Identification and validation of targets for cancer immunotherapy: from the bench-to-bedside

Chapter 12 in Novel Gene Therapy Approache

Application of the pMHC array to characterise tumour antigen specific T cell populations in leukaemia patients at disease diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients

Correlation between the detection of specific T cell populations using flow cytometry and pMHC arrays.

<p>Negatively isolated CD8⁺ T cells from a normal donor who was pMHC-Flu M1 positive and CMV sero-negative were <b>(A)</b> labelled with anti-CD8-FITC (y-axis) and pMHC-(Flu or CMV)-SA-PE (x-axis) and analysed by flow cytometry. We showed that a minimum 0.7 x 10⁶ CD8⁺ cells (including controls) could be used to show that the sample had Flu M1⁺ specific T cells but not A2/CMV pp65 specific T cells by conventional flow cytometry. CD8-FITC (FL1-H) is shown on the x-axis and pMHC-SA-PE (FL2-H) staining on the y-axis. <b>(B)</b> 10⁶ CD8⁺ T cells/ml, from the same normal donor sample, were lipophillically dyed with DiD and incubated for 20 minutes at 37°C with a custom-made hydrogel slide. Unbound cells were washed away with warm X- VIVO. CD8⁺ T cells (shown stained red) are visible at the single cell level bound to the Flu tetramer, which is visualised by the 5% AlexaFluor532 conjugated to streptavidin (shown as a green spot), on the ProScanArray. Composites show the co-localisation of Flu-specific CD8⁺ T cells to the Flu M1 tetramer spot. Few if any T cells are bound to the CMV pp65 pMHC spot or the negative control random pMHC library (also tetramerised with 5% AF532-streptavidin in streptavidin). Limits of detection in both assays were ≤0.1% of the CD8⁺ population.</p

PASD1 expression in AML patient samples.

<p>Immunolabelling was used to detect PASD1 protein expression in leukaemia patient samples. Haematoxylin provides the blue background stain to allow differentiation of the cell nucleus from the cytoplasm. The brown precipitate indicates a positive reaction between secondary antibody and detection reagent, at the antigen site. In this image AML004, AML008 and AML014 immunolabelled cells are shown, AML015 was one of the samples which did not have demonstrable PASD1 expression. No primary antibody was used as a negative control and actin was used as a positive control for immunolabelling.</p

CD8⁺ T cells from leukaemia patients binding to pMHC spots on the pMHC array.

<p><b>(A + B)</b> SEM showing a CD8⁺ positive T cell bound at the site of a tetramer spot on the pMHC array; <b>(C)</b> a single pit on the pMHC array suggesting that T cells dig into the hydrogel when binding to pMHCs and subsequently detach.</p

Diagrammatic representation of the pMHC array.

<p>Lipophilically dyed CD8⁺ T cells (0.8–1.2 x 10⁶ per microarray) were incubated at 37°C in warm colourless X-VIVO 15 media with the pMHC array. Each pMHC spot contains 1ng of tetramer and each slide can hold up to 1,000 spots of pMHC.</p

pMHCs used on the array to detect virus and LAA-specific T cell populations within the peripheral blood CD8+ population of leukaemia patients.

<p>^aOriginal reference for the epitope</p><p>^b245V mutation of MHC class I as described by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140483#pone.0140483.ref030" target="_blank">30</a>]</p><p>^ca random selection of 6,000 peptides, generated as described in reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140483#pone.0140483.ref017" target="_blank">17</a>].</p><p>HLA: human leukocyte antigen; Mod.: modified; Mut.: mutated; NK: not known.</p><p>pMHCs used on the array to detect virus and LAA-specific T cell populations within the peripheral blood CD8+ population of leukaemia patients.</p

Direct comparison of epitope specific T cell detection by pMHC tetramers following negative CD8⁺ isolation by conventional techniques.

<p>CD8⁺ T cells were isolated from HLA-A*0201⁺, normal donor buffy coat using commercially available negative isolation kits. This normal donor was CMV sero-negative, pMHC-CMV pp65 negative and pMHC-Flu M1 positive. <b>(A)</b> 10⁶ cells per test were incubated with 1μg of anti-CD8-FITC antibody, the cells washed and analysed by flow cytometry using a FACScalibur^TM. Method 2 gave the highest number of CD8⁺ cells (FL1). <b>(B)</b> 10⁶ CD8 T cells isolated from the same normal donor by Method 1 or 2 were labelled with anti-CD8-FITC (y-axis) and pMHC-(Flu or CMV)-SA-PE (x-axis) and analysed by flow cytometry. We found that Method 2 StemSep negative isolation of CD8s provided the best separation of pMHC-labelled CD8 T cells, in this case with Flu M1 tetramers.</p