Impact of CD4 variants on leukocyte differentiation, function, and TCR signaling. (A) Flow cytometry following extracellular staining of T-blasts from one healthy donor (HD3) and patients (P1, P3–P6). Cells were gated as follows: CD20⁻CD3⁺TCRγδ⁻TCRαβ⁺CD8⁻. Y axis represents (top) CD4 D1mAb (RPAT4) or (bottom) CD4 D4mAb (EPR6855). X axis represents CD4 D3mAb (SK3). Red gate represents the CD4 D4mAb single positive cell population. (B) Flow cytometry following extracellular staining of cryopreserved PBMCs from three HDs and patients (P1–P3, P6, P7). Cells were gated as follows: CD20⁻CD3⁺TCRγδ⁻TCRαβ⁺CD8⁻. Y axis represents (top) CD4 D1mAb (RPAT4) or (bottom) CD4 D4 mAb (EPR6855). X axis represents CD4 D3mAb (SK3). Red gate represents the CD4 D4mAb single positive cell population. (C) ZAP70 phosphorylation after crosslink. Rested T-blasts from P7, incubated in the absence or presence of either biotinylated CD3 (OKT3) alone (plain line) or CD3 (OKT3) with CD4 (D4mAb) (dotted line), and crosslinked with streptavidin for 1, 2, and 5 min. The geometric mean of ZAP70 phosphorylation was assessed by flowcytometry. Y axis: the geometric mean of ZAP70 phosphorylation upon stimulation normalized by non-stimulated condition; x axis: time of stimulation (streptavidin only). (D) CD3⁺ T lymphocytes subset frequencies in HDs, heterozygous relatives, and patients (P1–P7). (E) MAIT (CD3⁺CD161⁺TCR-Vα7.2⁺) cell frequency in HDs, heterozygous relatives, and patients (P1–P7). (F) NKT cell (CD3⁺Vα24JαQ⁺non-MAIT) frequency in HDs, heterozygous relatives, and patients (P1–P7). (G) CD3⁺TCRαβ⁺CD8⁻CD127⁻CD25⁺) Treg-like cell frequencies in HDs, heterozygous relatives, and patients (P1–P7). (H) CD3⁺ TCRγδ⁺ and TCRαβ⁺ frequencies in HDs, heterozygous relatives, and patients (P1–P7). (I and J) CD3⁺TCRαβ⁺CD8⁺ (I) and CD3⁺TCRαβ⁺CD8⁻ (J) T cell subpopulation frequencies in HDs, heterozygous relatives and patients (P1–P7). TCM: T central memory; TEM: T effector memory; EMRA: CD45RA⁺ effector memory. Data are representative of at least two independent experiments.