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T helper lymphocyte- and monocyte-specific type I interferon (IFN) signatures in autoimmunity and viral infection-GSE51997

Purpose

his study demonstrates quantitative and qualitative differences between type I IFN signatures in autoimmunity and viral infection using purified CD4pos T cells and CD16pos- and CD16neg-monocyte subsets. We were able to discriminate between cell-specific viral response signatures and the pathogenically amplified IFN signatures observed in autoimmunity.

Hypothesis

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Experimental Design

Collection of cells from Systemic Lupus Erythematosus (SLE) patients: For CD4pos T cells, six patients with SLE (average age: 29.0 +/- 7.6) and four normal healthy donors (ND; 24.8 +/- 0.5) were recruited. For CD16neg monocytes, four patients with SLE (26.5 +/- 1.7) and four NDs (24.8 +/- 0.5) were recruited. For CD16pos monocytes, four patients with SLE (26.5 +/- 1.7) and three NDs (24.7 +/- 0.6) were recruited. All patients and NDs were female. The same NDs were examined before and after immunisation with yellow fever vaccine (YFV). Collection of cells from yellow-fever vaccinated individuals: ND were immunised with a vaccine against the wild-type YF virus, which is a single-stranded RNA virus without adjuvants. This vaccine consists of a live but attenuated strain of the yellow fever virus (YFV-17D). Based on its vaccination-associated clinical and serological manifestations, this immunisation can be regarded as a real viral infection. A total of 50 ml peripheral blood was taken 7 days after immunisation, when sufficient numbers of CD19pos/CD27high plasmablasts were detected by flow cytometry. Cell sorting: A total of 50 ml peripheral blood was collected in Vacutainer heparin tubes and erythrocytes were lysed in EL buffer. Subsequently, granulocytes were depleted using CD15-conjugated microbeads (MACS). The CD15-depleted fraction was stained with a CD14-fluorescein isothiocyanate (FITC) antibody, a CD16-APC-Cy7 antibody, a CD3-Vioblue antibody and a CD4-FITC antibody. Using a FACSAria cell sorter, CD4pos T cells, CD16neg monocytes and CD16pos monocytes were isolated with purities and viabilities of >97%. After sorting, the cells were immediately lysed with RLT buffer and frozen at -70 °C. Total RNA was isolated using an RNeasy mini kit, and quality control was ensured by Bioanalyser measurements.Total RNA was extracted using the RNeasy Mini kit. The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer and a NanoDrop ND-1000 spectrophotometer. Biotinylated complementary RNA (cRNA) was synthesized from 100 ng total RNA, using reagents as recommended in the technical manual from Affymetrix. Fifteen micrograms of fragmented cRNA of each sample were hybridized to HG-U133 plus 2.0 arrays. Hybridization was performed according to procedure 2 as described in the technical manual. Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.

Experimental Variables

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Controls

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Methods

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Additional Information
  • Kyogoku C, Smiljanovic B, Grün JR, Biesen R et al. Cell-specific type I IFN signatures in autoimmunity and viral infection: what makes the difference?PLoS One 2013;8(12):e83776. PMID: 24391825
  • Microarray
    Affymetrix HG-U133_Plus_2
    36 Samples Loaded: 36
    Human (Homo sapiens)
    T cells , Monocytes
    Autoimmune Diseases
    Sample Set Spreadsheet
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    Samples Preview
    Sample ID !Sample_title cell type blood donor flow cytometric cell sorting vaccination
    GSM1256958 SLE-patients; Group 1: CD4 T lymphocytes; active SLE 2 CD4 SLE patient CD3posCD4pos none
    GSM1256959 SLE-patients; Group 1: CD4 T lymphocytes; active SLE 4 CD4 SLE patient CD3posCD4pos none
    GSM1256960 SLE-patients; Group 1: CD4 T lymphocytes; active SLE 7 CD4 SLE patient CD3posCD4pos none
    GSM1256961 SLE-patients; Group 1: CD4 T lymphocytes; active SLE 8 CD4 SLE patient CD3posCD4pos none
    GSM1256962 SLE-patients; Group 1: CD4 T lymphocytes; active SLE 9 CD4 SLE patient CD3posCD4pos none
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    Sample ID !Sample Title Cell type Blood donor Flow cytometric cell sorting Vaccination Sample contiditions
    GSM1256958
    SLE-patients; Group 1: CD4 T lymphocytes; active SLE 2
    CD4
    SLE patient
    CD3posCD4pos
    none
    SLE_CD4
    GSM1256959
    SLE-patients; Group 1: CD4 T lymphocytes; active SLE 4
    CD4
    SLE patient
    CD3posCD4pos
    none
    SLE_CD4
    GSM1256960
    SLE-patients; Group 1: CD4 T lymphocytes; active SLE 7
    CD4
    SLE patient
    CD3posCD4pos
    none
    SLE_CD4
    GSM1256961
    SLE-patients; Group 1: CD4 T lymphocytes; active SLE 8
    CD4
    SLE patient
    CD3posCD4pos
    none
    SLE_CD4
    GSM1256962
    SLE-patients; Group 1: CD4 T lymphocytes; active SLE 9
    CD4
    SLE patient
    CD3posCD4pos
    none
    SLE_CD4
    GSM1256963
    SLE-patients; Group 1: CD4 T lymphocytes; active SLE 12
    CD4
    SLE patient
    CD3posCD4pos
    none
    SLE_CD4
    GSM1256964
    SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 1
    CD16 negative
    SLE patient
    CD15negCD14posCD16neg
    none
    SLE_CD16-
    GSM1256965
    SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 2
    CD16 negative
    SLE patient
    CD15negCD14posCD16neg
    none
    SLE_CD16-
    GSM1256966
    SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 3
    CD16 negative
    SLE patient
    CD15negCD14posCD16neg
    none
    SLE_CD16-
    GSM1256967
    SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 4
    CD16 negative
    SLE patient
    CD15negCD14posCD16neg
    none
    SLE_CD16-
    GSM1256968
    SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 1
    CD16 positive
    SLE patient
    CD15negCD14posCD16pos
    none
    SLE_CD16+
    GSM1256969
    SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 2
    CD16 positive
    SLE patient
    CD15negCD14posCD16pos
    none
    SLE_CD16+
    GSM1256970
    SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 3
    CD16 positive
    SLE patient
    CD15negCD14posCD16pos
    none
    SLE_CD16+
    GSM1256971
    SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 4
    CD16 positive
    SLE patient
    CD15negCD14posCD16pos
    none
    SLE_CD16+
    GSM1256972
    Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 54 before YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    none
    CD4
    GSM1256973
    Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 55 before YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    none
    CD4
    GSM1256974
    Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 56 before YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    none
    CD4
    GSM1256975
    Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 57 before YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    none
    CD4
    GSM1256976
    Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 54 seven days after YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    7 days after Yellow Fever vaccination
    CD4 after YFV
    GSM1256977
    Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 55 seven days after YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    7 days after Yellow Fever vaccination
    CD4 after YFV
    GSM1256978
    Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 56 seven days after YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    7 days after Yellow Fever vaccination
    CD4 after YFV
    GSM1256979
    Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 57 seven days after YFV
    CD4
    Healthy individual (ND)
    CD3posCD4pos
    7 days after Yellow Fever vaccination
    CD4 after YFV
    GSM1256980
    Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 54 before YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    none
    CD16-
    GSM1256981
    Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 55 before YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    none
    CD16-
    GSM1256982
    Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 56 before YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    none
    CD16-
    GSM1256983
    Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 57 before YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    none
    CD16-
    GSM1256984
    Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 54 seven days after YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    7 days after Yellow Fever vaccination
    CD16- after YFV
    GSM1256985
    Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 55 seven days after YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    7 days after Yellow Fever vaccination
    CD16- after YFV
    GSM1256986
    Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 56 seven days after YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    7 days after Yellow Fever vaccination
    CD16- after YFV
    GSM1256987
    Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 57 seven days after YFV
    CD16 negative
    Healthy individual (ND)
    CD15negCD14posCD16neg
    7 days after Yellow Fever vaccination
    CD16- after YFV
    GSM1256988
    Yellow fever vaccinated NDs; Group 8: CD16pos, resident monocytes; ND 55 before YFV
    CD16 positive
    Healthy individual (ND)
    CD15negCD14posCD16pos
    none
    CD16+
    GSM1256989
    Yellow fever vaccinated NDs; Group 8: CD16pos, resident monocytes; ND 56 before YFV
    CD16 positive
    Healthy individual (ND)
    CD15negCD14posCD16pos
    none
    CD16+
    GSM1256990
    Yellow fever vaccinated NDs; Group 8: CD16pos, resident monocytes; ND 57 before YFV
    CD16 positive
    Healthy individual (ND)
    CD15negCD14posCD16pos
    none
    CD16+
    GSM1256991
    Yellow fever vaccinated NDs; Group 9: CD16pos, resident monocytes; ND 55 seven days after YFV
    CD16 positive
    Healthy individual (ND)
    CD15negCD14posCD16pos
    7 days after Yellow Fever vaccination
    CD16+ after YFV
    GSM1256992
    Yellow fever vaccinated NDs; Group 9: CD16pos, resident monocytes; ND 56 seven days after YFV
    CD16 positive
    Healthy individual (ND)
    CD15negCD14posCD16pos
    7 days after Yellow Fever vaccination
    CD16+ after YFV
    GSM1256993
    Yellow fever vaccinated NDs; Group 9: CD16pos, resident monocytes; ND 57 seven days after YFV
    CD16 positive
    Healthy individual (ND)
    CD15negCD14posCD16pos
    7 days after Yellow Fever vaccination
    CD16+ after YFV

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