Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells.  However, unlike CD8 T cells, CD4 T cells – especially self-reactive cells – are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of their low frequencies, and low affinities of their T cell receptors to MHC-peptide complexes.  In our efforts to improve the sensitivity of MHC class II tetramers, we recently created next-generation tetramers, designated ‘dextramers’. The dextramer reagents were helpful tools to detect and study the functionalities of autoreactive CD4 T cells in several murine autoimmune disease models. These include experimental autoimmune encephalomyelitis induced with proteolipid protein 139-151, and myelin oligodendrocyte glycoprotein 35-55; and myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352 and coxsackievirus B3 (CVB).  Here, we report five key findings.  (i) dextramers and not tetramers, permitted us to enumerate the precursor frequencies of antigen-specific CD4 T cells ex vivo in the brain and heart by flow cytometry;  (ii) direct staining with dextramers was sufficient to detect antigen-reactive T cells in situ by confocal microscopy; (iii) using Myhc-α 334-352 dextramers, for the first time, we showed that CVB infection leads to the generation of pathogenic cardiac myosin-specific CD4 T cells; (iv) dextramers were powerful tools to derive antigen-specific T cell hybridomas including evaluation of T cell markers, T cell receptors and their vβ usage in a single step by flow cytometry; and (v) characterization of pathogenic potential of dextramer⁺ cells based on  cytokine-secretion and chemokine receptor expression led us to conclude that autoreactive T cells sensitized with antigens derived from different organ systems do not have common cytokine signatures.  The implications of these observations will be discussed.