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A time dependent gene expression level of C3, C3aR and CTSL in CD4+ T cells
Abstracts / Immunobiology 221 (2016) 1131–1225
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A time dependent gene expression level of C3, C3aR and CTSL in CD4+ T cells
A novel, multiplexed targeted mass spectrometry assay for quantification of complement factor H (CFH) variants and CFH-related proteins 1–5 in human plasma
Cecilie Bo Hansen 1,∗ , Anton Willer 1 , Hanne Vibeke Hansen Marquart 1 , Martin Kolev 2 , Claudia Kemper 2 , Peter Garred 1 1 Laboratory of Molecular Medicine, Department of Clinical Immunology, Faculty of Health and Medical Sciences, University Hospital of Copenhagen, 2200 Copenhagen, Denmark 2 Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King’s College London, Guy’s Hospital, London SE1 9RT, UK
The complement system plays a central role in the elimination of microbes and dying host cells, but recent literature illuminates that certain complement proteins are of importance in relation to cell growth and survival. Almost all immune cells – incl. T- and B-cells have receptors that can bind complement, and through complement receptors activate specific cellular processes. But complement molecules can also be activated intracellularly and it has been shown that particular complement molecules are of vital importance for the differentiation and function of specific lymphocyte populations such as T cells. Resting human CD4+ T cells contain intracellular stores of C3 and the protease cathepsin L (CTSL) in endosomal and lysosomal compartments, and CTSL cleaves C3 into C3a and C3b. Intracellular C3a can thus bind to the lysosomallocalized receptor, C3aR, and mediate T-cell survival. This auto- and paracrine interaction between the complement system and the T cell will consequently determine T cell function and activity. Previous research has observed an up-regulation of C3 mRNA in CD4+ T cells upon certain stimulation and activation regiments, but to our knowledge, never in a time dependent manner. In the present study, we activated purified healthy CD4+ T cells by antibody coupled beads (␣-CD2, ␣-CD3 and ␣-CD28) and observed the difference in C3, C3aR and CTSL mRNA expression level between activated and non-activated CD4+ T cells. We activated the T cells in the course of 4 days, and assessed the gene expression level at 24 h, 48 h, 72 h and 96 h. We tested the activation by flow cytometry to evaluate if we were truly inducing a proliferation. Our preliminary results show a noticeably higher mRNA expression of C3, C3aR and CTSL in non-activated CD4+ T cells compared to activated CD4+ T cells. The difference in mRNA levels was persistent throughout all 4 time points. We also observed a slight decrease in the mRNA levels of activated CD4+ T cells during the 4 day experiment, which leads to the notion that there might be a reversed scenario in the first 24 h of activation, with initial high mRNA levels in activated CD4+ T cells. It is possible that we observe the aftermath of a vigorous machinery of complement production and overturning. To further evaluate and elucidate the initial gene expression level of C3, C3aR and CTSL in CD4+ T cells, we will perform activation experiments within the first 24 h. http://dx.doi.org/10.1016/j.imbio.2016.06.169
1201
Richard D. Semba 1,∗ , Pingbo Zhang 1 , Min Zhu 2 , Minghui Geng-Spyropoulos 1 , Michelle Shardell 2 , Marta Gonzalez-Freire 2 , Gudny Eiriksdottir 3,4 , Vilmunder Gudnason 3,4 , Jennifer E. Van Eyk 5 , Luigi Ferrucci 2 1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2 National Institute on Aging, National Institutes of Health, Baltimore, MD, USA 3 Icelandic Heart Association, Reykjavik, Iceland 4 Department of Medicine, University of Iceland, Reykjavik, Iceland 5 Advanced Clinical BioSystems Research Institute, The Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA
Age-related macular degeneration is the leading cause of visual loss among adults aged 65 or older in developed countries. Two major variants in the complement factor H gene, tyrosine 402 to histidine (Y402H) and isoleucine 62 to valine (I62V), are strongly associated with the risk of age-related macular degeneration. CFH is encoded in the Regulator of Complement Activation gene cluster in chromosome 1q32, which also includes five complement factor-related (CFHR) proteins, CFHR1 to CFHR5, with high amino acid sequence homology to CFH. A common haplotype, with deletion of CFHR1 and CFHR3 (CNP147 deletion) is associated with a decreased risk of AMD. Because of the high sequence homology among CFH variants and CFHR1-5, the optimal approach for characterization of these proteoforms in plasma is through use of selected reaction monitoring (SRM). We describe a SRM assay to measure the plasma concentrations of CFH variants Y402, H402, I62, and V62, and CFHR1-5. Tryptic peptides unique to each protein were identified using PeptideCutter, NCBI BLAST, and UniProt/BLAST searches and further refinement through Skyline. Seven proteotypic peptides were selected for CFH and its four variants. Seven proteotypic peptides were selected for CFHR1-5. Natural and stable isotope standard (SIS) peptides were synthesized. For peptides containing methionine, both oxidized and unoxidized SIS peptides were used. All peptides were optimized and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer (5500 QTrap, Sciex). The final assay consisted of 36 peptides and 108 interference-free SRM transition ion pairs. Most peptides showed good linearity over 0.3–200 fmol/L concentration range. Plasma concentrations of CFH variants and CFHR1-5 were measured using the SRM assay in 344 adults. Plasma CFH concentrations (mean, SE in g/mL) by genotype were: YY402, II62 (170.1, 31.4), YY402, VV62 (188.8, 38.5), HH402, VV62 (144.0, 37.0), HY402, VV62 (164.2, 42.3), YY402, IV62 (194.8, 36.8), HY402, IV62 (181.3, 44.7). There were no individuals with HH402, II62 or HH402, IV62 genotype. Mean (SD) plasma concentrations of CFHR1-5 were 1.63 (0.04), 3.64 (1.20), 0.020 (0.001), 2.42 (0.18), and 5.49 (1.55) g/mL, respectively. This SRM assay should facilitate the study of the role of systemic complement and the risk of age-related macular degeneration in future studies. http://dx.doi.org/10.1016/j.imbio.2016.06.170
154
155
A time dependent gene expression level of C3, C3aR and CTSL in CD4+ T cells
A novel, multiplexed targeted mass spectrometry assay for quantification of complement factor H (CFH) variants and CFH-related proteins 1–5 in human plasma
Cecilie Bo Hansen 1,∗ , Anton Willer 1 , Hanne Vibeke Hansen Marquart 1 , Martin Kolev 2 , Claudia Kemper 2 , Peter Garred 1 1 Laboratory of Molecular Medicine, Department of Clinical Immunology, Faculty of Health and Medical Sciences, University Hospital of Copenhagen, 2200 Copenhagen, Denmark 2 Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King’s College London, Guy’s Hospital, London SE1 9RT, UK
The complement system plays a central role in the elimination of microbes and dying host cells, but recent literature illuminates that certain complement proteins are of importance in relation to cell growth and survival. Almost all immune cells – incl. T- and B-cells have receptors that can bind complement, and through complement receptors activate specific cellular processes. But complement molecules can also be activated intracellularly and it has been shown that particular complement molecules are of vital importance for the differentiation and function of specific lymphocyte populations such as T cells. Resting human CD4+ T cells contain intracellular stores of C3 and the protease cathepsin L (CTSL) in endosomal and lysosomal compartments, and CTSL cleaves C3 into C3a and C3b. Intracellular C3a can thus bind to the lysosomallocalized receptor, C3aR, and mediate T-cell survival. This auto- and paracrine interaction between the complement system and the T cell will consequently determine T cell function and activity. Previous research has observed an up-regulation of C3 mRNA in CD4+ T cells upon certain stimulation and activation regiments, but to our knowledge, never in a time dependent manner. In the present study, we activated purified healthy CD4+ T cells by antibody coupled beads (␣-CD2, ␣-CD3 and ␣-CD28) and observed the difference in C3, C3aR and CTSL mRNA expression level between activated and non-activated CD4+ T cells. We activated the T cells in the course of 4 days, and assessed the gene expression level at 24 h, 48 h, 72 h and 96 h. We tested the activation by flow cytometry to evaluate if we were truly inducing a proliferation. Our preliminary results show a noticeably higher mRNA expression of C3, C3aR and CTSL in non-activated CD4+ T cells compared to activated CD4+ T cells. The difference in mRNA levels was persistent throughout all 4 time points. We also observed a slight decrease in the mRNA levels of activated CD4+ T cells during the 4 day experiment, which leads to the notion that there might be a reversed scenario in the first 24 h of activation, with initial high mRNA levels in activated CD4+ T cells. It is possible that we observe the aftermath of a vigorous machinery of complement production and overturning. To further evaluate and elucidate the initial gene expression level of C3, C3aR and CTSL in CD4+ T cells, we will perform activation experiments within the first 24 h. http://dx.doi.org/10.1016/j.imbio.2016.06.169
1201
Richard D. Semba 1,∗ , Pingbo Zhang 1 , Min Zhu 2 , Minghui Geng-Spyropoulos 1 , Michelle Shardell 2 , Marta Gonzalez-Freire 2 , Gudny Eiriksdottir 3,4 , Vilmunder Gudnason 3,4 , Jennifer E. Van Eyk 5 , Luigi Ferrucci 2 1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2 National Institute on Aging, National Institutes of Health, Baltimore, MD, USA 3 Icelandic Heart Association, Reykjavik, Iceland 4 Department of Medicine, University of Iceland, Reykjavik, Iceland 5 Advanced Clinical BioSystems Research Institute, The Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA
Age-related macular degeneration is the leading cause of visual loss among adults aged 65 or older in developed countries. Two major variants in the complement factor H gene, tyrosine 402 to histidine (Y402H) and isoleucine 62 to valine (I62V), are strongly associated with the risk of age-related macular degeneration. CFH is encoded in the Regulator of Complement Activation gene cluster in chromosome 1q32, which also includes five complement factor-related (CFHR) proteins, CFHR1 to CFHR5, with high amino acid sequence homology to CFH. A common haplotype, with deletion of CFHR1 and CFHR3 (CNP147 deletion) is associated with a decreased risk of AMD. Because of the high sequence homology among CFH variants and CFHR1-5, the optimal approach for characterization of these proteoforms in plasma is through use of selected reaction monitoring (SRM). We describe a SRM assay to measure the plasma concentrations of CFH variants Y402, H402, I62, and V62, and CFHR1-5. Tryptic peptides unique to each protein were identified using PeptideCutter, NCBI BLAST, and UniProt/BLAST searches and further refinement through Skyline. Seven proteotypic peptides were selected for CFH and its four variants. Seven proteotypic peptides were selected for CFHR1-5. Natural and stable isotope standard (SIS) peptides were synthesized. For peptides containing methionine, both oxidized and unoxidized SIS peptides were used. All peptides were optimized and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer (5500 QTrap, Sciex). The final assay consisted of 36 peptides and 108 interference-free SRM transition ion pairs. Most peptides showed good linearity over 0.3–200 fmol/L concentration range. Plasma concentrations of CFH variants and CFHR1-5 were measured using the SRM assay in 344 adults. Plasma CFH concentrations (mean, SE in g/mL) by genotype were: YY402, II62 (170.1, 31.4), YY402, VV62 (188.8, 38.5), HH402, VV62 (144.0, 37.0), HY402, VV62 (164.2, 42.3), YY402, IV62 (194.8, 36.8), HY402, IV62 (181.3, 44.7). There were no individuals with HH402, II62 or HH402, IV62 genotype. Mean (SD) plasma concentrations of CFHR1-5 were 1.63 (0.04), 3.64 (1.20), 0.020 (0.001), 2.42 (0.18), and 5.49 (1.55) g/mL, respectively. This SRM assay should facilitate the study of the role of systemic complement and the risk of age-related macular degeneration in future studies. http://dx.doi.org/10.1016/j.imbio.2016.06.170