It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule but AescinIIB the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti-Tim-1 antibodies induced CD3 capping only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity AescinIIB with which AescinIIB it is engaged. We recently identified a new gene family encoding the T cell immunoglobulin mucin (Tim) proteins (1 2 The Tim family consists of eight genes in mouse (cDNA was cloned into the pDisplay vector (Invitrogen) and transfected into Chinese hamster ovary (CHO) and EL-4 cells using a previously described method (43). Anti-Tim-1 mAb 3B3 (rat IgG2a κ) was generated using mucinless Tim-1-Ig which contains the IgV domain only as immunogen (14). Anti-mouse Tim-1 mAb RMT1-10 (rat IgG2a κ) was generated by immunizing SD rats with full-length Tim-1-Ig that contained both IgV and mucin domains of Tim-1. Lymph node cells were then fused with P3U1 myeloma cells and cloned. The hybridomas were screened for binding to mouse Tim-1-transfected CHO cells but not the parental CHO cells. The specificity of the anti-Tim-1 antibodies were further determined by staining EL-4 and CHO cells transfected with different Tim family members. Induction and clinical evaluation of EAE. 8-12-wk-old female SJL mice were immunized subcutaneously in the flanks with an emulsion containing PLP139-151 (80 μg/mouse) and 4 mg/ml H37Ra extract (DIFCO) in CFA. Pertussis toxin (100 ng/mouse; List Biological Laboratories) was administered intravenously on days 0 and 2. Mice were intraperitoneally injected with 100 μg 3B3 RMT1-10 rIgG or PBS every other day from day 0 to 8. Mice were monitored and assigned grades for clinical signs of EAE using the following scoring system: 0 healthy; 1 limp tail; 2 impaired righting reflex or waddled gait; 3 S1PR1 hind limb paralysis; 4 total limb paralysis; 5 moribund or death. At different time points brains and spinal cords AescinIIB were removed and fixed in 10% phosphate-buffered formalin and examined histologically for numbers of inflammatory foci and demyelination. Proliferation assays and ELISA. Female SJL mice were immunized with PLP139-151/CFA and treated with anti-Tim-1 or control antibodies as described in the previous section. Mice were killed at the time of disease onset (on day 10 for 3B3 treatment and on day 14 for RMT1-10 treatment) and spleens were removed. Spleen cells were isolated and plated in round-bottomed 96-well plates (BD Biosciences) in culture medium with various concentrations of PLP139-151. After 48 h culture supernatants were removed for cytokine ELISA and cytokine production was measured by quantitative capture ELISA as previously described (43). Plates were pulsed for 16 h with 1 μCi [3H]thymidine per well. Proliferation was measured as counts per minute by using a Wallac Liquid Scintillation AescinIIB Counter (Perkin Elmer). IAs Tetramer staining and intracellular staining. IAs tetramers for PLP139-151 and TMEV70-86 were generated as previously described (17 28 TMEV tetramers were used as negative controls. Lymphocytes from spleen and lymph nodes of PLP139-151-immunized SJL mice treated with different antibody were cultured with 20 μg/ml of antigen for 5 d. Cells were purified by Ficoll-Hypaque density gradient centrifugation. After washing cells were incubated with the tetramers (30 μg/ml) for 3 h at 37°C followed by staining with anti-CD4-APC (clone RM4.5) and 7-amino-actinomycin D (7-AAD; BD Biosciences). Cells were acquired by using the FACSort flow cytometer (Becton Dickinson) and tetramer-positive cells were determined within the CD4+ population after gating the viable cells (7-AAD?). FACS data were analyzed with the CELLQUEST (Becton Dickinson) and FlowJo (Tree Star) programs. AescinIIB To determine the frequency of cytokine-producing cells Ficoll-purified cells were reactivated with 20 ng/ml PMA (Sigma- Aldrich) and.