Helper T cells down-regulate CD4 expression upon chronic stimulation giving rise to double-negative T cells

Double-negative T (DNT) cells are αβTCR⁺CD3⁺CD4^-CD8^-NK1.1^- cells that constitute a small but significant proportion of the αβTCR⁺ T cells. Their developmental pathway and pathological significance remain unclear. In the present study, we utilized chronic in vitro stimulation of CD4⁺ T cells to mimic immune hyper-activation of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus, conditions characterized by DNT cells accumulation. After approximately 4-5 rounds of stimulation, the CD3⁺CD4^- population became apparent. These cells did not express CD8, NK1.1, γδTCR, or B220, exhibited a highly proliferative effector phenotype, and were dependent on T cell receptor (TCR) stimulation for survival. Moreover, CD3⁺CD4^- cells expressed MHC class II-restricted αβTCR, indicative of their origin from a CD4⁺ T cell population. The results presented herein illustrate a novel method of DNT cell generation in vitro and suggest that immune hyper-activation could also be implicated in the genesis of the disease-associated DNT cells in vivo.