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. 2024 Apr 1;221(5):e20231044. doi: 10.1084/jem.20231044

Figure 3.

Figure 3.

Characterization of the T cell compartment in patients with CD4 deficiency. (A) Flow cytometry following extracellular staining of T-blasts from two healthy donors (HDs) and P2. 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 cell population expressing isoform 2 only. (B) Flow cytometry following extracellular staining of PBMCs from two HDs, P4, and P5. 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) Immunoblots with LCK and GAPDH. Top: direct-IP with LCK; bottom: LCK co-IP with CD4 D4mAb (EPR6855). Experiments were conducted in THP1, T-blasts from four healthy donors (HD1–HD4) and patients (P1–P7). (D) Calcium flux mobilization in T-blasts from HDs (black, n = 7) and patients (red, P1–P6) upon TCR activation assessed by flow cytometry. Y axis represents the frequency of responding cells (ratio indo 1AM). X axis represents the time of acquisition. Arrows represent stimulation. SA: streptavidin; Iono: ionomycin. Each data point represents an average of 3 s. (E) ZAP70 phosphorylation after crosslink. Rested T-blasts from HDs (n = 8, black) and patients (P1–P6, red), 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: geometric mean of ZAP70 phosphorylation upon stimulation normalized by non-stimulated condition; x axis: time of stimulation (streptavidin only). Statistical analysis by one-tailed parametric paired Student’s t test. *P < 0.05; **P < 0.01. (F) CD3+TCRαβ+ CD8− and CD8+ T cell frequencies in HDs (n = 18), heterozygous relatives (n = 3), and patients (P1–P6). (G) CD3+TCRαβ+CD8−CD4− (CD4 D3mAB) DN T cell frequencies in HDs (n = 18), heterozygous relatives (n = 3), and patients (P1–P6). (F and G) Statistical analysis by one-way ANOVA with multiple comparisons (Tukey). **P < 0.01; ****P < 0.0001. (H) CD38 and CD45 staining defining CD3+TCRαβ+ conventional DN (CD38−CD45+/− blue gate) and pathogenic (CD38−CD45+/−, red gate) cell frequencies in one representative HD, FAS-deficient patient, STAT3 GOF, and CD4 patients with CD4 mutations (P1–P6). Brown gates represent total CD4+ T cells, green gates represent total CD8+ T cells, gray gates represent total DN cells. Data are representative of at least two independent experiments. Source data are available for this figure: SourceData F3.