Defective T-cell activation caused by impairment of the TNF receptor 2 costimulatory pathway in common variable immunodeficiency

Defective TCR stimulation in patients with CVID is not corrected if T cells are preactivated. T cells were precultured with or without anti-CD3 mAb for 10 to 17 days and restimulated with anti-TCR/anti-CD28 mAbs for 5 days. IFN-γ release was determined by ELISA (A; bars = median). At the end of preculture, the percentage of CD4 and CD8 positive cells was determined by flow cytometry (B). Co, Controls.

Calcium influx through SOC channels is normal in patients with CVID. PBMCs were loaded, stained for CD4, and resuspended in calcium-free medium. Baseline calcium concentration was established in the presence of ethyleneglycol-bis(β-aminoethylether)-N,N,N′-N′-tetraacetic acid (EGTA). Thapsigargin was added to deplete intracellular calcium stores. After a further 10 minutes, calcium was added, and the increase in intracellular calcium represents influx through SOC channels. MFI, Mean fluorescence intensity.

Translocation of PKC-θ is normal in patients with CVID. PBMCs were stimulated for 15 minutes (PMA 10 ng/mL), lysed, and separated into a membrane and cytosolic fraction. PKC-θ, PKC-βI (loading control), and fyn (membrane control) were examined by Western blot. (A) One representative experiment, (B) individual results for membrane extracts, and (C) densitometric readings (means ± SEMs) of membrane extracts. med., Cells treated with culture medium alone.

TCR costimulation via TNF-RII is defective in patients with CVID. Precultured (A; means ± SEMs; n = 8) or freshly isolated nonadherent lymphocytes (B; 1 experiment of 3) were stimulated with anti-TCR ± TNF-α or TNF-RII mAbs. C, Means ± SEMs, ● = XLA patient 3, ▴= XLA patient 5, ▪ = IgG subclass deficiency, ♦ = IgM deficiency. dpm, Disintegrations per minute.

TCR/CD28-induced proliferation and IL-2 secretion but not TCR/CD28-induced TRAF1 expression requires concomitant TNF-RII triggering. T cells were stimulated with anti-TCR/CD28 mAbs in the presence or absence of anti–TNF-α or an isotype control mAb (1 ug/mL). Bars represent the means ± SEMs of 4 (A) or 3 (B; IL-2 release determined by ELISA) experiments. C, TRAF1: 1 representative Western blot experiment of 4. med., Cells treated with culture medium alone; dpm, disintegrations per minute.

TCR and TCR/CD28-induced TRAF1 expression is normal in patients, but TCR/TNF-RII–induced TRAF1 expression is impaired. T cells were stimulated for 3 days (isotype control mAb coated beads = cobeads) and Western blots for TRAF1 (B) and TRAF2 were performed. The densitometric readings for TRAF1 are given in A (means ± SEMs; insert = densitometry for TRAF2). A ratio for TRAF1 densitometry was calculated in C. lono, Lonomycin; Co, controls.