Analysis of T regulatory cells (Treg) and T effector cells Gja7 (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. and not CD4norm cells cause EAE after adoptive transfer to na?ve recipients. Based on expression of CD4 and CD25 we next identify two separable cell populations of CD4+ CUDC-101 cells in antigen activated LN cultures CD4highCD25+ and CD4normalCD25+. The CD4high cells proliferate in response to specific antigen and do CUDC-101 not express Foxp3 while the CD4normal cell population does not proliferate to antigen stimulation expresses the Foxp3 gene and are able to suppress mitogen induced T cell responses. Lastly we show that CD4high but not CD4normal cells express the inflammatory cytokines IFN-γ and IL-17. Taken together we demonstrate that high expression of CD4 is a reliable marker of antigen-specific EAE effector T cells and can be used to separate this cell population from Treg cells from in vitro cultures. 2 Materials and Method 2.1 Mice B10.PL and C57BL/6 (B6) mice were purchased from the Jackson Laboratory (Bar Harbor ME) and maintained in the Stanford Medical Center Department of Comparative Medicine or the Wayne State University Department of Laboratory Animal Research. Mice were used between 6 and 12 weeks of age. All study related protocols were approved either by the Stanford University or Wayne State University animal investigation committees prior to performing the studies. 2.2 Antigens MOG peptide p35-55 (MEVGWYRPSFSRVVHLYRNGK) and MBP peptide Ac1-11 (AcASQKRPSQRHG) was synthesized by Genemed Synthesis (South San Fransisco CA) and purity confirmed by HPLC. 2.3 Immunization Groups of 3-5 mice were immunized subcutaneously at four sites in the flanks with 50 μl (each site) of an emulsion containing IFA plus 10 mg/ml heat killed H37RA (Difco Laboratories Inc Detroit MI) plus 200 μg/mouse antigen (MOG35-55 or MBPAc1-11) suspended in an equal volume of Dulbecco’s PBS. 2.4 In vitro culture Eight to ten days after immunization draining inguinal and axillary lymph nodes were removed and single cell suspensions prepared. LN cells were activated with the priming antigen (50 ug/ml) 8 x 106 cells/well in 24-well flat-bottom plates in EAE medium at 37° C supplemented with 5% CO2. EAE CUDC-101 media consisted of RPMI 1640 supplemented with 2 mM L-Glutamine penicillin/streptomycin nonessential amino acids sodium pyruvate and 10 mM hepes buffer (Gibco Laboratories Grand Island NY) 50 mM 2-ME (Sigma Chemical Co. St. Louis MO) and 10% FCS. For long term cultures cells were allowed to “rest” for 10 days without media addition. Cells were then restimulated as follows: T cells were harvested and cultured (2 x 106 cells) with APC’s (2.5 x 107 syngeneic irradiated spleen cells) and antigen (50 ug/ml) in T25 flasks in 10 ml EAE media. 2.5 FACS analysis For studies culture with the same antigen for four days. After FACS sorting or culture cells were washed and resuspended in PBS. Viable cells were assayed by tryphan blue exclusion counting. Cells were then resuspended to appropriate volumes for transfer to na?ve irradiated (500R) recipient mice which received the indicated number of viable cells by tail vein injection in 200 ul sterile PBS. Recipient mice were examined daily for clinical signs of disease and were graded according to the following scale: 0-no abnormality; 1-loss of tail tonicity; 2-paralysis in a single hind limb; 3-dual hind limb paralysis; 4-paralysis involving the forelimbs; 5-moribund; 6-death. CUDC-101 Subcutaneous injections of normal saline were administered to all animals losing significant body weight (> 20%). EAE was induced in B10.PL mice in a similar manner except that the antigen used was MBP Ac1-11 and recipient mice were not irradiated. After thirty days those mice that did not succumb to disease were challenged with a sub-encephalitogenic dose of antigen (50 ug/mouse) in CFA and the mice examined for signs of disease for the next sixty days. Disease was charted using the following criteria 1 average disease grade: average disease of those mice which succumbed to disease; 2) average day of onset: average day of onset of those mice which succumbed to disease; 3) incidence of disease: number of mice.