The activation requirements of autoreactive CD4⁺ T-cells were investigated in GAD65-specific HLA-DR0401–restricted clones derived from a diabetic patient using major histocompatibility complex (MHC) class II tetramers (TMrs) as stimulating agents. Despite the fact that TMrs loaded with an immunodominant-altered GAD peptide (TMr-GAD) bound a limited number of T-cell receptors, they were capable of efficiently delivering activation signals. These signals ranged from the early steps of phospholipase C (PLC)-γ₁ phosphorylation and Ca²⁺ mobilization to more complex events, such as CD69 upregulation, cytokine mRNA transcription and secretion, and proliferation. All the effects triggered by TMr-GAD were dose dependent. On the contrary, [³H]-thymidine incorporation decreased at high TMr-GAD concentrations because of activation-induced cell death (AICD) after initial proliferation. Lower-avidity clones (as defined by TMr-GAD binding) were less sensitive to activation as well as less susceptible to AICD compared with higher-avidity clones. Induction of apoptosis is a potential immunomodulatory target for therapeutic applications of MHC class II multimers, but the relative resistance of low-avidity T-cells may limit its benefits.