Transcriptome of myelin oligodendrocyte glycoprotein (MOG)-reactive CD4 T cells and control CD4 T cells from draining lymp nodes and spleens of mice during experimental autoimmune encephalomyelitis (EAE). Transcriptome of myelin oligodendrocyte glycoprotein (MOG)-reactive CD4 T cells and control CD4 T cells from draining lymp nodes and spleens of mice during experimental autoimmune encephalomyelitis (EAE)

MOG-reactive CD4 T cells were isolated from draining lymph nodes (dLN) and spleen of C57BL6/N female mice on day 10 after subcutaneous immunization with MOG(35-55) emulsified in Complete Freund’s adjuvant (CFA). Some mice received the CpG-B-1826 (50 microg; TCCATgACgTTCCTgACgTT; TIB MOLBIOL Syntheselabor GmbH), which was added to the MOG(35-55)/CFA mixture before emulsification. To isolate MOG-reactive CD4 T cells, dLN and spleens were harvested from mice on day 10 post-immunization, and single cell suspensions were re-stimulated for 5 hours using a mixture of MOG(35-55) (20 microg/ml), anti-CD40, anti-CD28, and anti-CD40L-PE (all from a commercial kit – Miltenyi Biotec – cat number 130-093-129). After 5 hours, CD4-positive cells were enriched magnetically by MACS using anti-CD4 microbeads (Miltenyi Biotec GmbH, cat. 130-049-201, clone L3T4), and subsequently subjected to FACS sorting to isolate CD4+CD40L+ and CD4+CD40L- cells after staining with anti-CD4 (clone RM4-5, conjugated to Pacific Blue), including a bin channel for CD8-positive cells (clone 53-6.7) and a marker for staining dead cells (NHPO). Sorted CD4+CD40L+ and CD4+CD40L- cells were then quickly checked for purity and directly lysed in 350 microlitre RLT buffer containing 1% beta-mercaptoethanol (Invitrogen). Purity was >95%. Mice were 6-10 weeks of age at the time of immunization. The goal was to identify genes differentially expressed between MOG-reactive CD4 T cells in mice treated or not with CpG-B, and to characterize the transcriptome of autoreactive CD4 T cells in draining lymph nodes and spleen on day 10 after immunization. Overall design: The experiments include a total of 15 arrays, each corresponding to a distinct biological sample. There are 3 arrays (3 samples) for CD4+CD40L+ cells from the dLN of control EAE mice. There are 3 arrays (3 samples) for CD4+CD40L+ cells from the dLN of CpG-treated EAE mice. There are 3 arrays (3 samples) for CD4+CD40L+ cells from the spleen of control EAE mice. There are 3 arrays (3 samples) for CD4+CD40L+ cells from the spleen of CpG-treated EAE mice. There are 3 arrays (3 samples) for CD4+CD40L- cells from the dLN of control EAE mice.