Characterization of Autoreactive CD4+ T cell Hybridomas

Autoimmune diseases are diverse disorders arising from abnormal cellular and humoral responses to self-antigens in the immune system. However, the underlying cause of autoimmunity remains unclear. In this study, ready-made autoreactive CD4+ T cell hybridomas (containing autoreactive CD4+ lymphocytes from scurfy TCRmini mice with BWα−β− thymoma line) were cloned and their antigen receptors (TCRs) were characterized to decipher the specificity of autoreactive TCRs. This approach was used to understand the mechanisms of autoreactivity in the immune system and assist in redirecting abnormal CD4+ T cells present in the peripheral repertoire of healthy individuals for therapeutic advantage. Some hybridoma clones expressed CD4+ and Vα2+ TCRs with unique amino acid sequences and they upregulated Nur77GFP expression upon autologous DCs stimulation and a stronger expression of Nur77GFP in the presence of aCD3 stimulation. Our data suggests that these activated hybridoma clones TCRs have an elevated binding affinity for self-peptides/MHCII complex and are autoreactive

Recognition of synthetic polyanionic ligands underlies “spontaneous” reactivity of Vγ1 γδTCRs

Abstract Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1-containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate-like αβ T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.</jats:p