The tumor-associated antigen RHAMM (HMMR/CD168) is expressed by monocyte-derived dendritic cells and presented to T cells

We formerly demonstrated that vaccination with Wilms' tumor 1 (WT1)-loaded autologous monocyte-derived dendritic cells (mo-DCs) can be a well-tolerated effective treatment in acute myeloid leukemia (AML) patients. Here, we investigated whether we could introduce the receptor for hyaluronic acid-mediated motility (RHAMM/HMMR/CD168), another clinically relevant tumor-associated antigen, into these mo-DCs through mRNA electroporation and elicit RHAMM-specific immune responses. While RHAMM mRNA electroporation significantly increased RHAMM protein expression by mo-DCs, our data indicate that classical mo-DCs already express and present RHAMM at sufficient levels to activate RHAMM-specific T cells, regardless of electroporation. Moreover, we found that RHAMM-specific T cells are present at vaccination sites in AML patients. Our findings implicate that we and others who are using classical mo-DCs for cancer immunotherapy are already vaccinating against RHAMM

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

<p>Abstract</p> <p>Background</p> <p>Bone marrow-derived stromal cells (MSC) are attractive targets for <it>ex vivo </it>cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord.</p> <p>Results</p> <p>First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While <it>in vivo </it>EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, <it>in vivo </it>NT3 mRNA expression by hMSC-NT3 cells and <it>in vivo </it>EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed <it>in vivo </it>decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants.</p> <p>Conclusion</p> <p>In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing <it>in vivo</it>.</p

Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple <it>in vitro </it>stimulations of CD8⁺T cells with peptide-pulsed autologous dendritic cells

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.</p> <p>Results</p> <p>In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E7_11–20peptide in order to induce an <it>in vitro </it>CD8⁺T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8⁺T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8⁺T cells in 5/5 healthy donors.</p> <p>Conclusion</p> <p>We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response <it>in vitro</it>. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses <it>in vitro</it>.</p

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord-4

<p><b>Copyright information:</b></p><p>Taken from "Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord"</p><p>http://www.biomedcentral.com/1472-6750/7/90</p><p>BMC Biotechnology 2007;7():90-90.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2225398.</p><p></p>ed slides pre-and post-LCM. (B) Upper graph showing the level of transgenic EGFP mRNA transcripts/1000 copies GAPDH in hMSC-EGFP cell cultures (CELLS, n = 1), on transplanted hMSC-EGFP cells at day 1 post-transplantation (DAY 1, n = 1), and on transplanted hMSC-EGFP cells at day 7 post-transplantation (DAY 7, n = 1). Lower graph showing the level of transgenic EGFP mRNA transcripts/1000 copies GAPDH in hMSC-NT3-EGFP cell cultures (CELLS, n = 1), on transplanted hMSC-NT3-EGFP cells at day 1 post-transplantation (DAY 1, n = 1), and on transplanted hMSC-NT3-EGFP cells at day 7 post-transplantation (DAY 7, n = 1)

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord-0

<p><b>Copyright information:</b></p><p>Taken from "Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord"</p><p>http://www.biomedcentral.com/1472-6750/7/90</p><p>BMC Biotechnology 2007;7():90-90.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2225398.</p><p></p>pulations. CMV: Cytomegalovirus immediate early promotor + enhancer. EGFP: enhanced green fluorescent protein. pA: SV40 early mRNA polyadenylation signal. NT3: neurothrophin-3. IRES: internal ribosome entry site. (B) Representative standard PCR and RT-PCR analysis on DNA and mRNA isolated from the different genetically modified hMSC populations used in this study (see numbers below pictures) indicating the presence of transgenic EGFP and/or NT3 DNA and mRNA sequences. M: length marker. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. (C) Representative real-time RT-PCR analysis on mRNA isolated from the different genetically modified hMSC populations used in this study (see numbers below pictures) showing quantitative differences in the level of transgenic EGFP and/or NT3 mRNA transcripts/1000 copies GAPDH; nd: no data available. (D) Representative ELISA measurement on supernatant samples from the different genetically modified hMSC populations used in this study (see numbers below pictures) showing quantitative differences in the level of NT3 secretion in picogram/10cells/24 hours. (E) Representative flow cytometric analysis of EGFP expression by hMSC-EGFP and hMSC-NT3-EGFP populations showing quantitative differences in the level of transgenic EGFP protein expression. SSC: side scatter

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord-1

<p><b>Copyright information:</b></p><p>Taken from "Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord"</p><p>http://www.biomedcentral.com/1472-6750/7/90</p><p>BMC Biotechnology 2007;7():90-90.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2225398.</p><p></p>d week 1–4). First row: hematoxylin-eosin (HE) staining indicating localisation and general appearance of transplantation site. Second row: direct EGFP fluorescence indicating the presence or absence of EGFP positive hMSC-EGFP cell transplants. Third row: immuno-histochemical staining for EGFP indicating the presence or absence of EGFP positive hMSC-EGFP cell transplants. Fourth row: immuno-histochemical staining for CD68 indicating macrophage infiltration into the transplantation site. All slides were examined using a conventional light/fluorescence microscope and digital pictures were taken under magnification as indicated. Representative pictures were chosen from multiple hMSC-EGFP cell transplanted spinal cords analysed for day 1 (n = 2), week 1 (n = 2), week 2 (n = 2), week 3 (n = 2), and week 4 (n = 6) post-transplantation. (B) Molecular and histological assessment of hMSC-EGFP cell transplant survival in rat spinal cord under systemic immune suppression (subcutaneous 10 mg/kg/day cyclosporin A) at four time points post transplantation (day 1 and week 1–3). DAY 1: Real-time RT-PCR analysis on mRNA isolated from hMSC-EGFP cell transplanted spinal cords on day 1 post-transplantation (n = 2) indicating the presence of EGFP mRNA transcripts in spinal cord following hMSC-EGFP cell transplantation. WEEK 1: direct EGFP fluorescence and immuno-histochemical staining for EGFP indicating the presence of EGFP positive hMSC-EGFP cell transplants on week 1 post-transplantation. Representative pictures were chosen from multiple hMSC-EGFP cell transplanted spinal cords analysed 1 week post-transplantation (n = 5). WEEK 2: HE staining, direct EGFP fluorescence and immuno-histochemical staining for EGFP indicating the presence of EGFP positive hMSC-EGFP cell transplants on week 2 post-transplantation. Representative pictures were chosen from multiple hMSC-EGFP cell transplanted spinal cords analysed 2 weeks post-transplantation (n = 3). WEEK 3: Standard PCR and RT-PCR analysis on DNA and mRNA isolated from hMSC-EGFP cell-transplanted spinal cords 3 weeks post-transplantation indicating the presence of EGFP DNA sequences and EGFP mRNA transcripts in spinal cord following hMSC-EGFP cell transplantation

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord-2

<p><b>Copyright information:</b></p><p>Taken from "Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord"</p><p>http://www.biomedcentral.com/1472-6750/7/90</p><p>BMC Biotechnology 2007;7():90-90.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2225398.</p><p></p>spinal cords on day 1 post-transplantation (n = 2) and on week 1 post-transplantation (n = 2) demonstrating the absence of detectable exogenous NT3 mRNA transcripts. SC: spinal cord