Furthermore, NFMlacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG<sub>35-55</sub> Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM<sub>18-30</sub>-specific T cells in the CNS were predominantly reactive to MOG<sub>38-49</sub> The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response.