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Sa.24. TCR-β Fingerprinting in Single T-Cells; a New High Throughput Method
Abstracts
Sa.23. The Role of IL-7 in the Induction of Regulatory T-Cells After Campath-1H. Vicki Robertson, Amanda Cox, Sara A. Thompson, Joanne Jones, D. Alastair Compston, Alasdair Coles. Neuroscience, University of Cambridge, Cambridge, United Kingdom. Regulatory T-cells (Tregs) are involved in the maintenance of peripheral tolerance, the control of non-self effector responses, and have a critical role in the prevention of autoimmune disease. The most widely studied population of Tregs are considered to be naturally occurring, and express both CD4 + CD25 high and the transcription factor FoxP3. Treatment of Multiple Sclerosis (MS) patients with alemtuzumab (Campath-1H), a monoclonal antibody therapy which induces a profound and prolonged lymphopaenia, results in an enrichment of putative Tregs one to three months post-treatment. In contrast to the suppressive activity of Tregs from healthy controls, CD4 + CD25 high cells, isolated from MS patients, have been reported to have impaired suppressive function. Here we describe the development of a functional Treg assay, which uses the fluorescent dye Carboxy fluoroscein diacetate succinimidyl ester (CFSE) to measure the suppressive activity of Tregs from MS patients pre- and post-treatment with Campath-1H, compared to healthy controls. As well as a putative enrichment of Tregs, a significant increase in serum interleukin-7 (IL-7) was also observed post Campath-1H treatment. IL-7 has been reported to induce the transient expression of high levels of CD25 on CD4+ cells, without the cells acquiring suppressor function. Spiking healthy control human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of recombinant human IL-7, results in an increase in the percentage of CD4+ cells that express high levels of CD25. We investigate whether the putative Tregs, observed post-Campath-1H treatment of MS patients, are transiently expressing CD25 (indicative of a Treg phenotype) as a result of an increase in serum IL-7, or whether these cells have the ability to suppress T effector cell proliferation. doi:10.1016/j.clim.2006.04.255
Sa.24. TCR-B Fingerprinting in Single T-Cells: a New High Throughput Method. Rajneesh Srivastava, Dun Zhou, Verena Grummel, Friederike Vogel, Sabine Cepok, Hans-Peter Hartung, Bernhard Hemmer. Department of Neurology, Neuroimmunology Group, Heinrich-Heine University, Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany. Analysis of the rearranged T-cell receptor genes, in particular the more polymorphic TCR-h gene provides a molecular fingerprinting of each T-cell. The TCR fingerprinting is essential to investigate T-cells responses in human autoimmune disease,infection & cancer.Since the TCR-h locus contains 55 variable(V) region gene segments,multiple assays have been necessary to determine TCR-h rearrangement of individual T-cell.We established a semi nested rt-PCR method for single T-cell analysis with
S113 two set of degenerate primers covering 76% and 24% of the TCR-Vh genes respectively. The specificity of the approach was validated by screening cDNAs obtained from T-cell clones (TCC) with defined TCR-h rearrangement. We applied the method successfully to profile TCR-h rearrangement on laser microdissected and sorted T-cell from tissue and body fluids respectively. Concomitant analysis of other gene transcripts allowed determining phenotype and function of TCR-h defined single T-cells. Our fast, cost-efficient and high throughput approach will facilitate studies on T-cells responses in human disease. doi:10.1016/j.clim.2006.04.256
Sa.25. In Vitro Correlates of in Vivo Rapamycin Therapy in Patients with Multiple Sclerosis. Jacqueline Slavik,1 Vissia Viglietta,1 Katarzyna Bourcier,2 Irina Grinberg,1 Samia Khoury,1 David Hafler.1 1 Neurology, Brigham and Women’s Hospital, Boston, MA; 2 Immune Tolerance Network, University of California, San Francisco, CA. Rapamycin has been shown to improve autoimmune diseases in animal models such as EAE, the NOD mouse and adjuvant-induced arthritis, and to help prevent autoimmune rejection of transplanted islet cells in humans. We embarked on a Phase I/II, open-label, pilot trial to evaluate the safety and efficacy of RapamuneR in patients with Multiple Sclerosis (MS) (see also Viglietta et al). Previously, we found that depending on the strength of the signal delivered to the T-cell via both the T-cell receptor (TcR) and CD28, CD8+ T-cells are capable of rapamycin-resistant proliferation. Rapamycin-resistant proliferation was associated with bcl-xL induction and p27kip1 downregulation. A major goal here was to examine the in vitro correlates of in vivo rapamycin therapy in patients with the autoimmune disease MS, in order to determine the in vivo mechanism of drug action. We first compared how in vitro rapamycin affects the proliferation of PBMC from patients with MS and control subjects. We found that the proliferative response to T-cell receptor (TcR) crosslinking was inhibited to a greater extent in the cells from patients with MS when compared to cells from controls. We next assessed the ex vivo proliferative response to TCR crosslinking as well as the antigenspecific proliferation of PBMC from patients with MS over the course of rapamycin therapy to examine how in vivo rapamycin therapy affects T-cell function measured ex vivo. We serially examined the ex vivo responsiveness of PBMC to TCR cross-linking during treatment with rapamycin and found that over time, response to these non-specific stimuli increased slightly in the four patients that remained clinically stable during treatment. This increase appeared to be independent of both strengths of signal delivered via the TCR and CD28 and of additional in vitro rapamycin exposure and was not evident in the four patients that failed therapy. doi:10.1016/j.clim.2006.04.257
Sa.23. The Role of IL-7 in the Induction of Regulatory T-Cells After Campath-1H. Vicki Robertson, Amanda Cox, Sara A. Thompson, Joanne Jones, D. Alastair Compston, Alasdair Coles. Neuroscience, University of Cambridge, Cambridge, United Kingdom. Regulatory T-cells (Tregs) are involved in the maintenance of peripheral tolerance, the control of non-self effector responses, and have a critical role in the prevention of autoimmune disease. The most widely studied population of Tregs are considered to be naturally occurring, and express both CD4 + CD25 high and the transcription factor FoxP3. Treatment of Multiple Sclerosis (MS) patients with alemtuzumab (Campath-1H), a monoclonal antibody therapy which induces a profound and prolonged lymphopaenia, results in an enrichment of putative Tregs one to three months post-treatment. In contrast to the suppressive activity of Tregs from healthy controls, CD4 + CD25 high cells, isolated from MS patients, have been reported to have impaired suppressive function. Here we describe the development of a functional Treg assay, which uses the fluorescent dye Carboxy fluoroscein diacetate succinimidyl ester (CFSE) to measure the suppressive activity of Tregs from MS patients pre- and post-treatment with Campath-1H, compared to healthy controls. As well as a putative enrichment of Tregs, a significant increase in serum interleukin-7 (IL-7) was also observed post Campath-1H treatment. IL-7 has been reported to induce the transient expression of high levels of CD25 on CD4+ cells, without the cells acquiring suppressor function. Spiking healthy control human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of recombinant human IL-7, results in an increase in the percentage of CD4+ cells that express high levels of CD25. We investigate whether the putative Tregs, observed post-Campath-1H treatment of MS patients, are transiently expressing CD25 (indicative of a Treg phenotype) as a result of an increase in serum IL-7, or whether these cells have the ability to suppress T effector cell proliferation. doi:10.1016/j.clim.2006.04.255
Sa.24. TCR-B Fingerprinting in Single T-Cells: a New High Throughput Method. Rajneesh Srivastava, Dun Zhou, Verena Grummel, Friederike Vogel, Sabine Cepok, Hans-Peter Hartung, Bernhard Hemmer. Department of Neurology, Neuroimmunology Group, Heinrich-Heine University, Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany. Analysis of the rearranged T-cell receptor genes, in particular the more polymorphic TCR-h gene provides a molecular fingerprinting of each T-cell. The TCR fingerprinting is essential to investigate T-cells responses in human autoimmune disease,infection & cancer.Since the TCR-h locus contains 55 variable(V) region gene segments,multiple assays have been necessary to determine TCR-h rearrangement of individual T-cell.We established a semi nested rt-PCR method for single T-cell analysis with
S113 two set of degenerate primers covering 76% and 24% of the TCR-Vh genes respectively. The specificity of the approach was validated by screening cDNAs obtained from T-cell clones (TCC) with defined TCR-h rearrangement. We applied the method successfully to profile TCR-h rearrangement on laser microdissected and sorted T-cell from tissue and body fluids respectively. Concomitant analysis of other gene transcripts allowed determining phenotype and function of TCR-h defined single T-cells. Our fast, cost-efficient and high throughput approach will facilitate studies on T-cells responses in human disease. doi:10.1016/j.clim.2006.04.256
Sa.25. In Vitro Correlates of in Vivo Rapamycin Therapy in Patients with Multiple Sclerosis. Jacqueline Slavik,1 Vissia Viglietta,1 Katarzyna Bourcier,2 Irina Grinberg,1 Samia Khoury,1 David Hafler.1 1 Neurology, Brigham and Women’s Hospital, Boston, MA; 2 Immune Tolerance Network, University of California, San Francisco, CA. Rapamycin has been shown to improve autoimmune diseases in animal models such as EAE, the NOD mouse and adjuvant-induced arthritis, and to help prevent autoimmune rejection of transplanted islet cells in humans. We embarked on a Phase I/II, open-label, pilot trial to evaluate the safety and efficacy of RapamuneR in patients with Multiple Sclerosis (MS) (see also Viglietta et al). Previously, we found that depending on the strength of the signal delivered to the T-cell via both the T-cell receptor (TcR) and CD28, CD8+ T-cells are capable of rapamycin-resistant proliferation. Rapamycin-resistant proliferation was associated with bcl-xL induction and p27kip1 downregulation. A major goal here was to examine the in vitro correlates of in vivo rapamycin therapy in patients with the autoimmune disease MS, in order to determine the in vivo mechanism of drug action. We first compared how in vitro rapamycin affects the proliferation of PBMC from patients with MS and control subjects. We found that the proliferative response to T-cell receptor (TcR) crosslinking was inhibited to a greater extent in the cells from patients with MS when compared to cells from controls. We next assessed the ex vivo proliferative response to TCR crosslinking as well as the antigenspecific proliferation of PBMC from patients with MS over the course of rapamycin therapy to examine how in vivo rapamycin therapy affects T-cell function measured ex vivo. We serially examined the ex vivo responsiveness of PBMC to TCR cross-linking during treatment with rapamycin and found that over time, response to these non-specific stimuli increased slightly in the four patients that remained clinically stable during treatment. This increase appeared to be independent of both strengths of signal delivered via the TCR and CD28 and of additional in vitro rapamycin exposure and was not evident in the four patients that failed therapy. doi:10.1016/j.clim.2006.04.257