F.35. Contrasting Responses of Human Microglia and Monocytes to Dendritic Cell-inducing Conditions
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(MS); however, after accounting for the effect of the well validated HLA DRB1*1501 susceptibility allele, none of these SNPs has a significant residual asociation to susceptibility to MS. doi:10.1016/j.clim.2008.03.145 F.34. Changes in the CD8dimCD4- Cell Population Parallel Disease Activity in Treated Multiple Sclerosis Subjects Philip De Jager, 1 Pia Kivisäkk, 1 Xinli Hu, 2 Dulce Soler-Ferran, 3 Elena Izmailova, 3 Carmeline O'Brien, 3 David Hafler, 1 Howard Weiner, 1 Samia Khoury. 11 Brigham and Women's Hospital, Boston, MA; 2 Broad Institute, Boston, MA; 3 Millenium Pharmaceuticals, Cambridge, MA As part of the Multiple Sclerosis Registry, we have acquired an ex vivo immunological profile from the whole blood of healthy subjects and subjects with remitting relapsing multiple sclerosis (RRMS). We have previously reported that the frequency of CD8dim-CD4- cells is reduced in 38 untreated RRMS subjects (mean 5.1%) when compared to 32 healthy control subjects (mean 7.5%) (P = 0.00020). Using independent subjects from the Com- prehensive Longitudinal Investigation of MS at the Brigham & Women's Hospital, we have replicated this result seen in 16 untreated RRMS subjects (mean 5.1%) when compared to 18 healthy control subjects (mean 7.3%)(P = 0.0016). The frequency of this population is also reduced in 11 untreated subjects with a clinically isolated demyelinating syndrome (mean 5.5%). Furthermore, this CD8dim-CD4- cell population may be a barometer of disease activity in treated MS subjects. As others have shown with daclizumab and interferon b treatment, we demonstrate a normal- ization of CD8dim-CD4- frequency (mean 7.4%) in 10 subjects treated with either glatiramer acetate or inter- feron b who are clinically and radiographically stable after at least 12 months of treatment. Finally, we see a significant drop in the frequency of this cell population to a mean of 4.6% in 9 subjects who display evidence of disease activity after at least 3 months of treatment with either agent (P = 0.011). The analysis of validation data in an additional 100 treatment responders and 100 treatment nonresponders is ongoing and will be presented at the conference. doi:10.1016/j.clim.2008.03.146 F.35. Contrasting Responses of Human Microglia and Monocytes to Dendritic Cell-inducing Conditions Caroline Lambert, 1 Julie Desbarats, 1 Nathalie Arbour, 2 Jack Antel. 11 McGill University, Montreal, QC, Canada; 2 Research Centre of the Centre Hospitalier de l'Universite de Montreal, Montreal, QC, Canada Microglia are myeloid resident cells of the central nervous system (CNS) able to adapt their activation response to a variety of stimuli. However, their specific contribution in triggering and sustaining local immune responses in the human CNS is a subject of debate. We examined the potential of both fetal and adult human microglia to acquire dendritic cell (DC) properties in response to GM-CSF and IL-4 differentiation followed by maturation with LPS, a conventional protocol used to differentiate monocytes into mature DCs. Using this protocol, monocytes became MHC class II hi , CD14 lo , CD209 + , CD1a + , CD83 + DCs. Likewise, fetal and adult microglia became CD14 lo and CD209 + but remained CD1a - , CD83 - and decreased their levels of MHC class II. Differentiated microglia produced significant amounts of IL-10 and TNF but no detectable IL-12 following LPS maturation, in contrast to monocytes, which also produced IL-12. As expected, monocyte-derived DCs had a marked ability to promote CD4 T cell proliferation in an allogeneic mixed leukocyte reaction. Fetal microglia stimulated with GM-CSF+IL-4+LPS had an improved ability to promote CD4 T cell proliferation but to a lower extent than DCs. In contrast, similarly treated adult microglia had a decreased ability to stimulate CD4 T cell proliferation compared with their untreated counterparts. Our data indicate that fetal and adult microglia treated with DC-inducing conditions in vitro adopt an analogous phenotype that is not character- istic of mature DCs. Moreover, adult microglia appear to have modulated their intrinsic properties, possibly as part of their developmental process, to promote tolerance in the CNS. doi:10.1016/j.clim.2008.03.147 F.36. Introduction of a Cell-based Assay Against Native Aquaporin-4—High Specificity and Sensitivity for Neuromyelitis Optica Sudhakar Kalluri, 1 Til Menge, 2 Rajneesh Srivastava, 1 Bruce Cree, 3 Bernhard Hemmer. 11 Technical University Munich, Munich, Germany; 2 Heinrich-Heine University, Dusseldorf, Germany; 3 University of California San Francisco, San Francisco, CA Neuromyelitis optica (NMO) is chronic demyelinating autoimmune disorder of the central nervous system with partial patho-etiological overlap to multiple sclerosis (MS). Recently, an antibody response against the waterchannel protein aquaporin-4 (AQP-4) has been established as diag- nostic biomarker for NMO. Current assay methodologies consist of indirect immunofluorescence and radioimmuno- precipitation assays, respectively. Here we report on a cell- based flow cytometry assay; the human glioblastoma cell line LN18 was stably transfected using a lentiviral vector to overexpress human AQP 4. Expression was monitored by immunocytochemistry and western blotting of cell lysates. Antibody reactivity was assessed in 56 human samples (17 healthy controls (HC), 22 MS, 17 NMO) by flow cytometry at 1/100 dilutions. Results were expressed as median fluores- cence intensities (MFI) corrected for background binding to the sham-transfected cell line. Samples were deemed positive if the MFI was above a cut-off (MFI (HC) + 10*SD). 15 NMO samples were positive (median MFI 534, range 66- 2880), but no MS HC samples, respectively (p b 0.05; ANOVA for multiple MFI comparison); median endpoint titers were S54 Abstracts
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Transcript of F.35. Contrasting Responses of Human Microglia and Monocytes to Dendritic Cell-inducing Conditions
(MS); however, after accounting for the effect of the wellvalidated HLA DRB1*1501 susceptibility allele, none of theseSNPs has a significant residual asociation to susceptibility toMS.
doi:10.1016/j.clim.2008.03.145
F.34. Changes in the CD8dimCD4- Cell PopulationParallel Disease Activity in Treated MultipleSclerosis SubjectsPhilip De Jager,1 Pia Kivisäkk,1 Xinli Hu,2 DulceSoler-Ferran,3 Elena Izmailova,3 Carmeline O'Brien,3 DavidHafler,1 Howard Weiner,1 Samia Khoury.1 1Brigham andWomen's Hospital, Boston, MA; 2Broad Institute, Boston,MA; 3Millenium Pharmaceuticals, Cambridge, MA
As part of the Multiple Sclerosis Registry, we haveacquired an ex vivo immunological profile from the wholeblood of healthy subjects and subjects with remittingrelapsing multiple sclerosis (RRMS). We have previouslyreported that the frequency of CD8dim-CD4- cells isreduced in 38 untreated RRMS subjects (mean 5.1%) whencompared to 32 healthy control subjects (mean 7.5%)(P=0.00020). Using independent subjects from the Com-prehensive Longitudinal Investigation of MS at the Brigham& Women's Hospital, we have replicated this result seen in16 untreated RRMS subjects (mean 5.1%) when comparedto 18 healthy control subjects (mean 7.3%)(P=0.0016). Thefrequency of this population is also reduced in 11untreated subjects with a clinically isolated demyelinatingsyndrome (mean 5.5%). Furthermore, this CD8dim-CD4- cellpopulation may be a barometer of disease activity intreated MS subjects. As others have shown with daclizumaband interferon b treatment, we demonstrate a normal-ization of CD8dim-CD4- frequency (mean 7.4%) in 10subjects treated with either glatiramer acetate or inter-feron b who are clinically and radiographically stable afterat least 12 months of treatment. Finally, we see asignificant drop in the frequency of this cell populationto a mean of 4.6% in 9 subjects who display evidence ofdisease activity after at least 3 months of treatment witheither agent (P=0.011). The analysis of validation data inan additional 100 treatment responders and 100 treatmentnonresponders is ongoing and will be presented at theconference.
doi:10.1016/j.clim.2008.03.146
F.35. Contrasting Responses of Human Microglia andMonocytes to Dendritic Cell-inducing ConditionsCaroline Lambert,1 Julie Desbarats,1 Nathalie Arbour,2 JackAntel.1 1McGill University, Montreal, QC, Canada; 2ResearchCentre of the Centre Hospitalier de l'Universite deMontreal, Montreal, QC, Canada
Microglia are myeloid resident cells of the centralnervous system (CNS) able to adapt their activationresponse to a variety of stimuli. However, their specificcontribution in triggering and sustaining local immune
responses in the human CNS is a subject of debate. Weexamined the potential of both fetal and adult humanmicroglia to acquire dendritic cell (DC) properties inresponse to GM-CSF and IL-4 differentiation followed bymaturation with LPS, a conventional protocol used todifferentiate monocytes into mature DCs. Using thisprotocol, monocytes became MHC class IIhi, CD14lo,CD209+, CD1a+, CD83+ DCs. Likewise, fetal and adultmicroglia became CD14lo and CD209+ but remained CD1a-,CD83- and decreased their levels of MHC class II.Differentiated microglia produced significant amounts ofIL-10 and TNF but no detectable IL-12 following LPSmaturation, in contrast to monocytes, which also producedIL-12. As expected, monocyte-derived DCs had a markedability to promote CD4 T cell proliferation in an allogeneicmixed leukocyte reaction. Fetal microglia stimulated withGM-CSF+IL-4+LPS had an improved ability to promote CD4 Tcell proliferation but to a lower extent than DCs. Incontrast, similarly treated adult microglia had a decreasedability to stimulate CD4 T cell proliferation compared withtheir untreated counterparts. Our data indicate that fetaland adult microglia treated with DC-inducing conditions invitro adopt an analogous phenotype that is not character-istic of mature DCs. Moreover, adult microglia appear tohave modulated their intrinsic properties, possibly as partof their developmental process, to promote tolerance inthe CNS.
doi:10.1016/j.clim.2008.03.147
F.36. Introduction of a Cell-based Assay AgainstNative Aquaporin-4—High Specificity andSensitivity for Neuromyelitis OpticaSudhakar Kalluri,1 Til Menge,2 Rajneesh Srivastava,1 BruceCree,3 Bernhard Hemmer.1 1Technical University Munich,Munich, Germany; 2Heinrich-Heine University, Dusseldorf,Germany; 3University of California San Francisco,San Francisco, CA
Neuromyelitis optica (NMO) is chronic demyelinatingautoimmune disorder of the central nervous system withpartial patho-etiological overlap to multiple sclerosis (MS).Recently, an antibody response against the waterchannelprotein aquaporin-4 (AQP-4) has been established as diag-nostic biomarker for NMO. Current assay methodologiesconsist of indirect immunofluorescence and radioimmuno-precipitation assays, respectively. Here we report on a cell-based flow cytometry assay; the human glioblastoma cellline LN18 was stably transfected using a lentiviral vector tooverexpress human AQP 4. Expression was monitored byimmunocytochemistry and western blotting of cell lysates.Antibody reactivity was assessed in 56 human samples (17healthy controls (HC), 22 MS, 17 NMO) by flow cytometry at1/100 dilutions. Results were expressed as median fluores-cence intensities (MFI) corrected for background binding tothe sham-transfected cell line. Samples were deemedpositive if the MFI was above a cut-off (MFI (HC) + 10*SD).15 NMO samples were positive (median MFI 534, range 66-2880), but no MS HC samples, respectively (pb0.05; ANOVAfor multiple MFI comparison); median endpoint titers were
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