Pascale Hubert

Idiopathic CD4 lymphocytopenia: clinical and immunologic characteristics and follow-up of 40 patients.

AbstractIdiopathic CD4 T lymphocytopenia (ICL) is a rare and severe condition with limited available data. We conducted a French multicenter study to analyze the clinical and immunologic characteristics of a cohort of patients with ICL according to the Centers for Disease Control criteria.We recruited 40 patients (24 female) of mean age 44.2 ± 12.2 (19–70) years. Patients underwent T-lymphocyte phenotyping and lymphoproliferation assay at diagnosis, and experiments related to thymic function and interferon (IFN)-&ggr; release by natural killer (NK) cell were performed. Mean follow-up was 6.9 ± 6.7 (0.14–24.3) years. Infectious, autoimmune, and neoplastic events were recorded, as were outcomes of interleukin 2 therapy.In all, 25 patients had opportunistic infections (12 with human papillomavirus infection), 14 had autoimmune symptoms, 5 had malignancies, and 8 had mild or no symptoms. At the time of diagnosis, the mean cell counts were as follows: mean CD4 cell count: 127/mm3 (range, 4–294); mean CD8: 236/mm3 (range, 1–1293); mean CD19: 113/mm3 (range, 3–547); and mean NK cell count: 122/mm3 (range, 5–416). Most patients had deficiency in CD8, CD19, and/or NK cells. Cytotoxic function of NK cells was normal, and patients with infections had a significantly lower NK cell count than those without (p = 0.01). Patients with autoimmune manifestations had increased CD8 T-cell count. Proliferation of thymic precursors, as assessed by T-cell rearrangement excision circles, was increased. Six patients died (15%). CD4 T-cell count <150/mm3 and NK cell count <100/mm3 were predictors of death.In conclusion, ICL is a heterogeneous disorder often associated with deficiencies in CD8, CD19, and/or NK cells. Long-term prognosis may be related to initial CD4 and NK cell deficiency.

Analysis by flow cytometry of tyrosine-phosphorylated proteins in activated T-cell subsets on whole blood samples

Tyrosine phosphorylation of cellular proteins is a critical early event in the T-cell activation process induced by the antigenic peptide or monoclonal antibodies specific for the CD3 T-cell receptor (TCR) complex. Phosphoproteins are currently detected by Western blotting experiments or, recently, by labelling intracellular proteins with an antiphosphotyrosine monoclonal antibody and flow cytometric analysis (Farahi Far et al.: Cytometry 15:327-334, 1994; Vuillier et al.: J Immunol Methods 185:43-56, 1995). Here, we describe improvements of these latter methods in order to study selectively the CD3-TCR signaling pathway of patients with immunodeficiency diseases or lymphopenia. This new technique quantifies tyrosine-phosphorylated proteins in in vitro-activated T-cell subsets directly on whole blood samples. The stimulation of the CD3-TCR complex induces a specific and significant increase in the phosphotyrosine fluorescence intensity in both CD4 and CD8 subpopulations. The simplicity and the good reproducibility of this method make it particularly convenient for laboratory routine evaluation, and the use of very small volumes of blood is well adapted to the study of immunodepressed patients. Moreover, this technique allows the detection of early molecular defects of the CD3-TCR signal-transduction pathway.

Priming of CD2-induced p62Dok tyrosine phosphorylation by CD3 in Jurkat T cells.

T lymphocyte activation is triggered through the CD3‐TCR complex or the CD2 molecule. Beside common biochemical events, we previously showed that a 62‐kDa protein associated with PLCγ‐1 and p21RasGAP was specifically tyrosine phosphorylated after CD2 stimulation in Jurkat T cells. We demonstrated here that it was identical to p62Dok, a docking protein highly phosphorylated in human chronic myelogenous leukemia cells and in murine abl‐transformed B cells. Mainly, we showed that p62Dok tyrosine phosphorylation was strengthened by the functional interplay between CD3 and CD2. Primary stimulation of Jurkat cells via CD3 suppressed most of the subsequent CD2‐dependent phosphorylation events, except p62Dok tyrosine phosphorylation, which was on the contrary strongly increased. Kinetic studies indicated that a short treatment with anti‐CD3 was sufficient to amplify the CD2‐induced tyrosine phosphorylation of p62Dok. By contrast, CD2‐induced PLCγ‐1 tyrosine phosphorylation and calcium response progressively diminished. Finally, enhanced amounts of tyrosine phosphorylated p62Dok were recruited to p21RasGAP and PLCγ‐1 after CD2 stimulation in CD3‐activated cells. CD3 stimulation is known to enhance CD2 avidity for its ligand and to induce the binding of the CD2AP protein to the CD2 cytoplasmic tail. Our results suggest that the CD3‐TCR complex rapidly primes the CD2 pathway to activate one of its specific components, p62Dok.