Iron selectively induces proliferation of Vδ2⁺ T cells in HDs, represses ALP transcription in primary tissues and cell lines, and iron overload is associated with the same phenotype in thalassemia (Th) as in individuals with HH. (A) Fe²⁺ spiked into HD PBMCs at concentrations observed in HH (10-100 μM) dose dependently and selectively stimulates Vδ2⁺ T-cell proliferation (supplemental Figure 3). HH Vδ2⁺ T cells have a significant lower replication index (fold expansion of responding Vδ2⁺ T cells) and a significantly lower expansion index (fold expansion of whole Vδ2⁺ cell population) than HD Vδ2⁺ T cells. (B) Fe2⁺-induced proliferation (n = 5) can be abrogated by BTN3A1-blocking antibody 103.2 in HD (open bars) (n = 5). (C) Fe²⁺ potently represses ALP transcription in primary tissues and derived cell lines. RNA was isolated 3 days after exposure to titrated Fe²⁺, allowing a meaningful chronological timeframe for transcriptional repression and measuring resulting proliferation of Vδ2Vg9 (analyzed at day 7). (D) Patients with Th major (HFE-independent iron overload n = 26) exhibit same phenotypic abnormalities in the γδ T-cell compartment as individuals with HH (C282Y n = 22, compound n = 11) compared with healthy individuals (n = 30). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant.