Artyomov, Maxim N.

Calvier, Laurent

Cravens, Petra D.

Cross, Anne H.

Doelger, Richard

Eagar, Todd N.

Edelson, Brian T.

Esaulova, Ekaterina

Forsthuber, Thomas G.

Herz, Joachim

Hussain, Rehana Z.

Loof, Nicolas

Manouchehri, Navid

Stüve, Olaf

Wu, Gregory F.

English

Digital Commons@Becker

  1   Supplementary Information for CD11c+CD88+CD317+ myeloid cells are critical mediators of persistent CNS autoimmunity  Navid Manouchehri1, Rehana Z. Hussain1, Petra D. Cravens1, Ekaterina Esaulova2, Maxim N. Artyomov2, Brian T. Edelson2, Gregory F. Wu2,3, Anne H. Cross3, Richard Doelger1, Nicolas Loof4, Todd N. Eagar5, Thomas G. Forsthuber6, Laurent Calvier7,8, Joachim Herz7,8,9,10, Olaf Stüve1,11  Author Affiliations: 1Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, TX, U.S.A. 2Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, U.S.A. 3Department of Neurology, Washington University School of Medicine, St. Louis, MO, U.S.A. 4The Moody Foundation Flow Cytometry Facility (N.L.), Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, U.S.A. 5Department of Pathology and Genomic Medicine, Methodist Hospital Houston, TX, U.S.A. 6Department of Biology, University of Texas at San Antonio, San Antonio, TX, U.S.A. 7Department of Molecular Genetics, University of Texas (UT) Southwestern Medical Center, Dallas, U.S.A. 8Center for Translational Neurodegeneration Research, UT Southwestern Medical Center, Dallas, U.S.A. 9Department of Neuroscience, UT Southwestern Medical Center, Dallas, U.S.A. 10Center for Neuroscience, Department of Neuroanatomy, Albert-Ludwigs-University, Freiburg, Germany 11Neurology Section, VA North Texas Health Care System, Medical Service, Dallas, TX, U.S.A.  Olaf Stüve, M.D., Ph.D  Email: olaf.stuve@utsouthwestern.edu  This PDF file includes:  Figures S1 to S3     2                         Supplementary Figure 1.  Flow cytometry study gating strategy.  Cells were gated according to morphology side scatter (SSC-A) vs forward scatter (FSC-A).  Doublets were excluded (FSC-A vs FSC-H and SSC-A vs SSC-H).  Live cells were selected using the viability dye. Flow cytometry density plots showing gating strategy used to identify CD11c+CD88+CD317+ cells (CD45+MHCII+D11c+CD88+CD317+), B cells (CD45+CD3-CD19+), CD4+ T cells (CD45+CD3+CD4+) and CD8+ T cells (CD45+CD3+CD8+).  Each sample contains a minimum of 50 x 103 live events.     3              Supplementary Figure 2.  The composition of leukocytes outside the CD11c+ lineage is not altered in CD11c.Cre+/-ITGA4fl/fl mice.  The mean ± standard deviation (SD) of leukocyte subsets (%) from total 50 x 103 recorded viable cells in different compartments, including (A-F) brain and spinal cord, (G-L) spleen and lymph node and (M-R) bone marrow and blood, is presented during naïve, acute and persistent clinical actively-induced experimental autoimmune encephalomyelitis (EAE) in CD11c.Cre+/-ITGA4fl/fl mice or C57BL/6 wild type (WT) controls (N =6 experimental animals per group; data show pooled analysis of all study cohorts).  There was no difference in frequency of cell types including CD45+, CD19+, CD4+ and CD8+ cells between the two groups during different stages of the disease (P value > 0.05).       4               Supplementary Figure 3: Characterization of CD11c+CD88+CD317+ cells and microglia using ex vivo flow cytometry. (A) CD45hi cells were not abundantly present in naïve CNS tissue.  (B) From CD45+ cells, (C) CD11c+MHCII+ and (D) CD88+CD317+ cells were selected.  Next, (E) CX3CR1+ cells were gated for, and selected for (F) expression of TMEM119+ or (J) the absent expression of TMEM119-.  (G) Iba-1+ cells were selected from TMEM119+cells.  (H, R) The majority of CX3CR1+TMEM119+Iba-1+ cells defined as parenchymal microglia localized in the CD45int gate.  (K) Iba-1- cells were selected from TMEM119-cells.  (L, S) The majority of CX3CR1+TMEM119-Iba-1- cells defined as non-microglia localized in the CD45hi gate.  (I) FewerCD45int microglia expressed CD86 compared to (M) CD45hi non-microglia.  (O, N) CD45+CD11c+CD88+ cells in the blood were mostly (P) CX3CR1+, but (Q) TMEM-119- and Iba-1- .   

CD11c+ CD88+ CD317+ myeloid cells are critical mediators of persistent CNS autoimmunity

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CD11c+ CD88+ CD317+ myeloid cells are critical mediators of persistent CNS autoimmunity

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