- Email: [email protected]
Su.97. Characterization of Cd4 T Lymphocyte Response to Anti-Hepatitis B Vaccination
S192 In addition, treatment with MGN-3 also significantly increased the production of cytokines TNF-a and IFN-g in human mononuclear cells (MNC) while maintaining cell viability, as compared to control cultures. Notably, incubation of MGN-3 alone with 31 types of bacteria did not affect bacterial growth, suggesting that MGN-3 does not have direct antibacterial activity, but rather acts to modulate phagocytic cellular function. These findings may have applications in the treatment of infections in the elderly. doi:10.1016/j.clim.2006.04.521
Su.95. Standardization of Cytokine Flow Cytometry Assays: NIH/NIAID, SeraCare and BD Biosciences Quality Assurance Program. Maria Jaimes,1 Janice Darden,2 Patricia D’Souza,2 Janet Lathey,3 Holden Maecker.1 1BD Biosciences, San Jose, CA; 2 Office for Policy in Clinical Research Operations, DAIDS/ NIAID/NIH, Bethesda, MD; 3Virology/Immunology, SeraCare BioServices, Gaithersburg, MD. Cytokine flow cytometry (CFC) is used to measure antigen-specific T-cell responses in settings such as experimental HIV vaccination. Standardization of CFC among different laboratories would provide a common endpoint assay for comparing the immunogenicity of different vaccine candidates in multiple clinical trials. For this purpose, a Quality Assurance Program has been developed in a collaborative effort between the NIH/ NIAID, SeraCare BioServices, and BD Biosciences, in which 12 sites worldwide are currently participating. A detailed common protocol was provided. In the first two rounds of this program, cryopreserved peripheral blood mononuclear cells (PBMC) from 4 CMV+ donors were tested for CD4+ and CD8+ IFNg and IL-2 responses after a 6 h stimulation using CEF or CMV pp65 peptide mixes. The peptide and antibody cocktails provided were lyophilized into 96-well plates since previous data suggest that lyophilized reagents provide increased standardization of the assay. Each site analyzed its data independently and also provided their raw data to a central site for analysis. In addition, each laboratory completed a questionnaire to evaluate details of how the experiment was performed (i.e. type of instrument used for acquisition, instrument settings, etc). After analysis of the data, the central site produced an individual report for each site. The first rounds of the program resulted in interlaboratory coefficients of variation (C.V.) ranging from 12—60%, for cytokine+ responses higher than 0.2% based on central analysis. Instrument settings (PMT voltages and compensation) and analysis (gating strategy) played a critical role in inter-laboratory variation. We expect to provide new ideas and tools that will help overcome these issues and also to extend this program to other laboratories using CFC in their research or clinical studies. doi:10.1016/j.clim.2006.04.522
Abstracts
Su.96. Changes in the Kinetics of Cellular Subpopulation After Treatment of Transfer Factor. A Case Report. Eduardo Bracamontes,4 Marisela Linares,1 Hector Perez,1 Sanjuanita Flores,1 Mayra Perez,2 Javier Barrientos,2 Abraham Portugues,2 Abelardo Monges,2 Yonathan Garfias,1 Raul Chavez,3 Iris Estrada,2 Sergio Estrada,2 Maria Carmen Jimenez-Martinez.1 1Research Unit, Institute of Opthalmology, Fundacion Conde Valenciana, Mexico, Mexico; 2Department of Immunology, ENCB, IPN, Mexico, Mexico; 3Department of Biochemistry, Laboratory of Immunology, Fac. Med., UNAM, Mexico, Mexico; 4 Otolaringology Service, Clinica Churubusco, ISSSTE, Mexico, Mexico. Transfer Factor (TF) are dialyzable leukocyte extracts and they are known as a modulators of immune response, for its immunological properties they have been used to treat patients with infectious diseases or with deficient Th1 response. The aim of this work was to evaluate T-cells subpopulations and cytokine production after therapy with TF. The clinical manifestations of EBV infection range from subclinical seroconversion to the syndrome of infectious mononucleosis (IM). Here we presented a women of 31 years-old, who were initially evaluated at the otolaryngology service with extreme tiredness declared for one week and who develop a classical syndrome of IM characterized by faringodinea, peripheric lymphadenopathies, amygdaline hypertrophy, lymphocytosis, neutropenia and thrombocytopenia with transiently hepatic disfunction. Material and Methods: The patient received 2 doses of TF and its immunologic status was evaluated before and after therapy. PBMC were isolated from peripheral blood and labeled with monoclonal antibodies against CD4, CD8 and CD57; in other step some cells were used to detect intracellular cytokines after policlonal stimuli (PMA/ionomycin). Results: Percentage of CD4+ T-cells before TF 23 F 2 vs 31 F 4 after TF (p = 0.03); percentage of CD8+ T-cells before 50 F 1 vs 61 F 2 after TF (p b 0.001); CD4+CD57+ T-cells before TF 3 F 1 vs 9 F 1 after TF (p b 0.001); CD8+CD57+ T-cells before TF 15 F 0.5 vs 19 F 1 after TF (p = 0.004). Cytokine production: IFN-g+ cells before TF 11 F 3 vs 22 F 4 after TF (p b 0.001).Conclusion: Our results showed changes in the kinetics of CD57 on CD4+ and CD8+ T-cells, as well as an increment in IFN-g+ T-cells this results open the possibility that TF could modulate immune response trough activation of NKT cells, however more studies are necesary to resolve this question. doi:10.1016/j.clim.2006.04.523
Su.97. Characterization of CD4 T Lymphocyte Response to Anti-Hepatitis B Vaccination. Muriel Stubbe, Nathalie Vanderheyde, Michel Goldman, Arnaud Marchant. Institute For Medical Immunology, University of Brussels, Charleroi, Belgium. CD4 T lymphocytes play a central role in orchestrating immune responses to vaccines. The differentiation of human CD4 T lymphocytes and their acquisition of effector functions in vivo remain poorly understood. In order to increase our understanding of this process, we studied the response
Abstracts
S193
of adult volunteers to the anti-hepatitis B vaccine (Engerix-B, GlaxoSmithKline). Volunteers were immunized at 0, 2 and 4 months and CD4 T-cell responses were measured at regular intervals up to 12 months after immunization. Antigen specific cells were detected ex vivo by flow cytometry following short time stimulation with the hepatitis B surface (Hbs) Ag and using intracellular CD154 as marker of activation. HBs-specific responses were detected in all subjects. The frequency of antigen specific cells (CD154+ cells/total CD4+ T-cells) peaked 2 weeks after the administration of the third vaccine dose (mean F SEM: 0.101 F 0.042%), declined thereafter and reached a plateau after 3 months. High proportions of activated cells (HLA-DR+, Ki67+) were detected following administration of the second and third vaccine doses and decreased thereafter. About 10% of Agspecific cells were Ki67+ during the memory phase of the response, suggesting intense homeostatic proliferation. During the memory phase, most HBs-specific cells were CCR7and CD45RO+ and about 50% were CD27-. This allowed the differentiation of three distinct populations: 10% of the cells were CCR7+/CD27+ (central memory), 50% were CCR7-/ CD27- (effector memory) and 40% were CCR7-/CD27+ suggesting an intermediate stage of differentiation. The majority of HBs-specific cells produced TNF-a, about 50% produced IL-2 and a minority produced IFN-g or IL-13. We conclude that the hepatitis B vaccine stimulates an heterogeneous population of CD4 T lymphocytes. The clonal basis and the functional consequences of this heterogeneity remain to be determined. doi:10.1016/j.clim.2006.04.524
Other Su.98. Defining a Role for FOXP3 in Human CD4+ T-Cells. Sarah Allan,1 Laura Passerini,2 Rosa Bacchetta,2 Mario Amendola,2 Luigi Naldini,2 Maria Grazia Roncarolo,2 Megan Levings,1. 1 Surgery, University of British Columbia, Vancouver, BC, Canada; 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy. Naturally occurring CD4+CD25+ regulatory T-cells (Tregs) maintain immune homeostasis by cell-contact-dependent suppression of effector T-cell responses. Based on gene knock-out and over-expression studies in mice, it has been demonstrated that a single transcription factor, FoxP3, has an essential role in the development and function of Tregs. In order to generate large numbers of antigen-specific Tregs for cellular therapy, we investigated the capacity of FOXP3 to drive the generation of human Tregs in vitro by transduction of naRve CD4+ T-cells. Surprisingly, retroviral-mediated ectopic expression of FOXP3 alone, or together with the FOXP3delta2 splice isoform, resulted in only a modest acquisition of suppressive activity. To test the hypothesis that strong TCR-mediated activation may render T-cells resistant to the pro-suppressive effects of FOXP3, we investigated whether lenti-viral mediated transfer of FOXP3 into resting naive CD4+ T-cells resulted in a more potent regulatory phenotype. Expression of FOXP3 in resting cells, however, also failed to induce a strong regulatory phenotype,
supporting the theory that optimal Treg development requires stimulation through the TCR. We also investigated the role of gamma-chain cytokines in the generation of suppressive FOXP3-transduced cells in vitro. Interestingly, cytokines in addition to IL-2 were found to affect the outcome of FOXP3-transduction. Moreover, different gamma-chain cytokines significantly influenced levels of FOXP3 expression in ex vivo Tregs. These data suggest that the cytokine microenvironment can influence the induction and/or maintenance of FOXP3 expression. Together, our data indicate that several factors influence FOXP3 expression and induction of suppressive activity in human T-cells, and that overexpression of FOXP3 alone in adult CD4+ T-cells is not sufficient to generate a population of cells that would be suitable for cellular therapy. doi:10.1016/j.clim.2006.04.525
Su.99. Human Antibodies for Immunotherapy Development Generated Via a Human B-Cell Hybridoma Technology. Jian Li,1 Tao Sai,1 Marc Berger,1 Qimin Chao,1 Diane Davidson,1 Gaurav Deshmukh,1 Brian Drozdowski,1 Wolfgang Ebel,1 Stephen Harley,1 Marianne Henry,1 Sara Jacob,1 Brad Kline,1 Ella Lazo,1 Frank Rotella,1 Kathryn Rudolf,1 Eric Routhier,1 Jeaneen Sage,1 Paul Simon,1 Jun Yao,1 Yuhong Zhou,1 Mani Kavuru,2 Tracey Bonfield,2 Mary Jane Thomassen,2 Philip M. Sass,1 Nicholas C. Nicolaides,1 Luigi Grasso.1 1Antibody Development, Morphotek Inc., Exton, PA; 2Pulmonary Function Lab, The Cleveland Clinic Foundation, Cleveland, OH. Current strategies for the production of therapeutic MAbs include the use of mammalian cell systems to recombinantly produce antibodies derived from mice bearing human immunoglobulin transgenes, humanization of rodent antibodies, or phage libraries. Generation of hybridomas secreting human MAbs has been previously reported however this approach has not been fully exploited for immunotherapy development. We have previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g. affinity, titers) of MAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be utilized to improve suboptimal hybridoma cells generated via ex-vivo immunization and immortalization of antigen-specific human B-cells for therapeutic antibodies development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human MAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting MAbs with high binding specificity and biological activity. One MAb with strong neutralizing activity against human GM-CSF was identified that is currently advanced to preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large scale manufacturing. doi:10.1016/j.clim.2006.04.526
Su.95. Standardization of Cytokine Flow Cytometry Assays: NIH/NIAID, SeraCare and BD Biosciences Quality Assurance Program. Maria Jaimes,1 Janice Darden,2 Patricia D’Souza,2 Janet Lathey,3 Holden Maecker.1 1BD Biosciences, San Jose, CA; 2 Office for Policy in Clinical Research Operations, DAIDS/ NIAID/NIH, Bethesda, MD; 3Virology/Immunology, SeraCare BioServices, Gaithersburg, MD. Cytokine flow cytometry (CFC) is used to measure antigen-specific T-cell responses in settings such as experimental HIV vaccination. Standardization of CFC among different laboratories would provide a common endpoint assay for comparing the immunogenicity of different vaccine candidates in multiple clinical trials. For this purpose, a Quality Assurance Program has been developed in a collaborative effort between the NIH/ NIAID, SeraCare BioServices, and BD Biosciences, in which 12 sites worldwide are currently participating. A detailed common protocol was provided. In the first two rounds of this program, cryopreserved peripheral blood mononuclear cells (PBMC) from 4 CMV+ donors were tested for CD4+ and CD8+ IFNg and IL-2 responses after a 6 h stimulation using CEF or CMV pp65 peptide mixes. The peptide and antibody cocktails provided were lyophilized into 96-well plates since previous data suggest that lyophilized reagents provide increased standardization of the assay. Each site analyzed its data independently and also provided their raw data to a central site for analysis. In addition, each laboratory completed a questionnaire to evaluate details of how the experiment was performed (i.e. type of instrument used for acquisition, instrument settings, etc). After analysis of the data, the central site produced an individual report for each site. The first rounds of the program resulted in interlaboratory coefficients of variation (C.V.) ranging from 12—60%, for cytokine+ responses higher than 0.2% based on central analysis. Instrument settings (PMT voltages and compensation) and analysis (gating strategy) played a critical role in inter-laboratory variation. We expect to provide new ideas and tools that will help overcome these issues and also to extend this program to other laboratories using CFC in their research or clinical studies. doi:10.1016/j.clim.2006.04.522
Abstracts
Su.96. Changes in the Kinetics of Cellular Subpopulation After Treatment of Transfer Factor. A Case Report. Eduardo Bracamontes,4 Marisela Linares,1 Hector Perez,1 Sanjuanita Flores,1 Mayra Perez,2 Javier Barrientos,2 Abraham Portugues,2 Abelardo Monges,2 Yonathan Garfias,1 Raul Chavez,3 Iris Estrada,2 Sergio Estrada,2 Maria Carmen Jimenez-Martinez.1 1Research Unit, Institute of Opthalmology, Fundacion Conde Valenciana, Mexico, Mexico; 2Department of Immunology, ENCB, IPN, Mexico, Mexico; 3Department of Biochemistry, Laboratory of Immunology, Fac. Med., UNAM, Mexico, Mexico; 4 Otolaringology Service, Clinica Churubusco, ISSSTE, Mexico, Mexico. Transfer Factor (TF) are dialyzable leukocyte extracts and they are known as a modulators of immune response, for its immunological properties they have been used to treat patients with infectious diseases or with deficient Th1 response. The aim of this work was to evaluate T-cells subpopulations and cytokine production after therapy with TF. The clinical manifestations of EBV infection range from subclinical seroconversion to the syndrome of infectious mononucleosis (IM). Here we presented a women of 31 years-old, who were initially evaluated at the otolaryngology service with extreme tiredness declared for one week and who develop a classical syndrome of IM characterized by faringodinea, peripheric lymphadenopathies, amygdaline hypertrophy, lymphocytosis, neutropenia and thrombocytopenia with transiently hepatic disfunction. Material and Methods: The patient received 2 doses of TF and its immunologic status was evaluated before and after therapy. PBMC were isolated from peripheral blood and labeled with monoclonal antibodies against CD4, CD8 and CD57; in other step some cells were used to detect intracellular cytokines after policlonal stimuli (PMA/ionomycin). Results: Percentage of CD4+ T-cells before TF 23 F 2 vs 31 F 4 after TF (p = 0.03); percentage of CD8+ T-cells before 50 F 1 vs 61 F 2 after TF (p b 0.001); CD4+CD57+ T-cells before TF 3 F 1 vs 9 F 1 after TF (p b 0.001); CD8+CD57+ T-cells before TF 15 F 0.5 vs 19 F 1 after TF (p = 0.004). Cytokine production: IFN-g+ cells before TF 11 F 3 vs 22 F 4 after TF (p b 0.001).Conclusion: Our results showed changes in the kinetics of CD57 on CD4+ and CD8+ T-cells, as well as an increment in IFN-g+ T-cells this results open the possibility that TF could modulate immune response trough activation of NKT cells, however more studies are necesary to resolve this question. doi:10.1016/j.clim.2006.04.523
Su.97. Characterization of CD4 T Lymphocyte Response to Anti-Hepatitis B Vaccination. Muriel Stubbe, Nathalie Vanderheyde, Michel Goldman, Arnaud Marchant. Institute For Medical Immunology, University of Brussels, Charleroi, Belgium. CD4 T lymphocytes play a central role in orchestrating immune responses to vaccines. The differentiation of human CD4 T lymphocytes and their acquisition of effector functions in vivo remain poorly understood. In order to increase our understanding of this process, we studied the response
Abstracts
S193
of adult volunteers to the anti-hepatitis B vaccine (Engerix-B, GlaxoSmithKline). Volunteers were immunized at 0, 2 and 4 months and CD4 T-cell responses were measured at regular intervals up to 12 months after immunization. Antigen specific cells were detected ex vivo by flow cytometry following short time stimulation with the hepatitis B surface (Hbs) Ag and using intracellular CD154 as marker of activation. HBs-specific responses were detected in all subjects. The frequency of antigen specific cells (CD154+ cells/total CD4+ T-cells) peaked 2 weeks after the administration of the third vaccine dose (mean F SEM: 0.101 F 0.042%), declined thereafter and reached a plateau after 3 months. High proportions of activated cells (HLA-DR+, Ki67+) were detected following administration of the second and third vaccine doses and decreased thereafter. About 10% of Agspecific cells were Ki67+ during the memory phase of the response, suggesting intense homeostatic proliferation. During the memory phase, most HBs-specific cells were CCR7and CD45RO+ and about 50% were CD27-. This allowed the differentiation of three distinct populations: 10% of the cells were CCR7+/CD27+ (central memory), 50% were CCR7-/ CD27- (effector memory) and 40% were CCR7-/CD27+ suggesting an intermediate stage of differentiation. The majority of HBs-specific cells produced TNF-a, about 50% produced IL-2 and a minority produced IFN-g or IL-13. We conclude that the hepatitis B vaccine stimulates an heterogeneous population of CD4 T lymphocytes. The clonal basis and the functional consequences of this heterogeneity remain to be determined. doi:10.1016/j.clim.2006.04.524
Other Su.98. Defining a Role for FOXP3 in Human CD4+ T-Cells. Sarah Allan,1 Laura Passerini,2 Rosa Bacchetta,2 Mario Amendola,2 Luigi Naldini,2 Maria Grazia Roncarolo,2 Megan Levings,1. 1 Surgery, University of British Columbia, Vancouver, BC, Canada; 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy. Naturally occurring CD4+CD25+ regulatory T-cells (Tregs) maintain immune homeostasis by cell-contact-dependent suppression of effector T-cell responses. Based on gene knock-out and over-expression studies in mice, it has been demonstrated that a single transcription factor, FoxP3, has an essential role in the development and function of Tregs. In order to generate large numbers of antigen-specific Tregs for cellular therapy, we investigated the capacity of FOXP3 to drive the generation of human Tregs in vitro by transduction of naRve CD4+ T-cells. Surprisingly, retroviral-mediated ectopic expression of FOXP3 alone, or together with the FOXP3delta2 splice isoform, resulted in only a modest acquisition of suppressive activity. To test the hypothesis that strong TCR-mediated activation may render T-cells resistant to the pro-suppressive effects of FOXP3, we investigated whether lenti-viral mediated transfer of FOXP3 into resting naive CD4+ T-cells resulted in a more potent regulatory phenotype. Expression of FOXP3 in resting cells, however, also failed to induce a strong regulatory phenotype,
supporting the theory that optimal Treg development requires stimulation through the TCR. We also investigated the role of gamma-chain cytokines in the generation of suppressive FOXP3-transduced cells in vitro. Interestingly, cytokines in addition to IL-2 were found to affect the outcome of FOXP3-transduction. Moreover, different gamma-chain cytokines significantly influenced levels of FOXP3 expression in ex vivo Tregs. These data suggest that the cytokine microenvironment can influence the induction and/or maintenance of FOXP3 expression. Together, our data indicate that several factors influence FOXP3 expression and induction of suppressive activity in human T-cells, and that overexpression of FOXP3 alone in adult CD4+ T-cells is not sufficient to generate a population of cells that would be suitable for cellular therapy. doi:10.1016/j.clim.2006.04.525
Su.99. Human Antibodies for Immunotherapy Development Generated Via a Human B-Cell Hybridoma Technology. Jian Li,1 Tao Sai,1 Marc Berger,1 Qimin Chao,1 Diane Davidson,1 Gaurav Deshmukh,1 Brian Drozdowski,1 Wolfgang Ebel,1 Stephen Harley,1 Marianne Henry,1 Sara Jacob,1 Brad Kline,1 Ella Lazo,1 Frank Rotella,1 Kathryn Rudolf,1 Eric Routhier,1 Jeaneen Sage,1 Paul Simon,1 Jun Yao,1 Yuhong Zhou,1 Mani Kavuru,2 Tracey Bonfield,2 Mary Jane Thomassen,2 Philip M. Sass,1 Nicholas C. Nicolaides,1 Luigi Grasso.1 1Antibody Development, Morphotek Inc., Exton, PA; 2Pulmonary Function Lab, The Cleveland Clinic Foundation, Cleveland, OH. Current strategies for the production of therapeutic MAbs include the use of mammalian cell systems to recombinantly produce antibodies derived from mice bearing human immunoglobulin transgenes, humanization of rodent antibodies, or phage libraries. Generation of hybridomas secreting human MAbs has been previously reported however this approach has not been fully exploited for immunotherapy development. We have previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g. affinity, titers) of MAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be utilized to improve suboptimal hybridoma cells generated via ex-vivo immunization and immortalization of antigen-specific human B-cells for therapeutic antibodies development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human MAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting MAbs with high binding specificity and biological activity. One MAb with strong neutralizing activity against human GM-CSF was identified that is currently advanced to preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large scale manufacturing. doi:10.1016/j.clim.2006.04.526