Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines

Abstract

Background Identification of human T‐helper cell subsets is possible by measurement of intracellular cytokines after coincubation of lymphocytes with phorbol myristate acetate (PMA), calcium ionophore, and brefeldin A for up to 20 h. However, exposure to PMA leads to internalization of membrane CD4 and to loss of resolution of the CD4+ cells. Detection of CD3+CD8‐ cells or preselection of CD4+ cells prior to stimulation is more cumbersome than direct measurement of CD4+ cells. We report the use of the Leu3a/Leu3b multiclone for the accurate determination of CD4 cells after PMA stimulation. Methods Peripheral blood lymphocytes were isolated from healthy normal donors and the proportion of CD3+ / CD4+ T cells was determined by flow cytometry before and after incubation with PMA, calcium ionophore, and brefeldin A for 20 h using a variety of anti‐CD4 monoclonal antibodies. Results The Leu3a/3b multiclone reagent was the only anti‐CD4 monoclonal antibody capable of resolving more than 98% of the initial CD4+ events after incubation with PMA. Conclusions The higher signal‐to‐noise ratio associated with Leu3a3b reagent, compared with other CD4‐specific antibodies available, allows the direct and accurate identification of the CD4 subset even after PMA treatment of cells. Cytometry 44:148–152, 2001. © 2001 Wiley‐Liss, Inc.

Patients with the most advanced rheumatoid arthritis remain with Th1 systemic defects after TNF inhibitors treatment despite clinical improvement

“…Because incubation with phorbol 12-myristate 23-acetate (PMA) triggers internalization and degradation of the CD4 molecule, which would affect the identification of Th1 (CD4+IL-17-IFN-γ+) and Th17 (CD4+IFN-γ-IL-17+) cells [9], CD4+ T cells were negatively separated before cytokine staining. CD4+ T cells purified by magnetic cell sorting (Miltenyi Biotec) were then stimulated with 25 ng/ml PMA and 1 μg/ml of ionomycin (Ion) (Sigma-Aldrich) in the presence of 10 μg/ml of brefeldin A (BFA, protein transport inhibitor) for 4 h at 37 °C in a humidified atmosphere containing 5 % CO 2 .…”

Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients

“…At least 100 000 cells were analysed, and the data were processed using the XL2 software. The analysis was direct for the co‐expression of CD8 and CD4 antigens with the various cytokines, as the expression of multiclone CD4 (Leu3a‐3b) has been previously shown to remain stable after incubation with phorbol esters (Hennesy et al , 2001). The lymphocytic population was initially defined in a forward (FSC) and side scatter (SSC) gate.…”

Differential Modulation of Surface and Intracellular Protein Expression by T Cells after Stimulation in the Presence of Monensin or Brefeldin A

“…The difficulty encountered by researchers analyzing intracellular cytokine expression by the CD4 subset is a direct result of the degree to which this marker is downregulated by PMA-ION stimulation. To circumvent this problem, various approaches have been suggested, including the analysis of CD8 Ϫ cells instead of CD4 ϩ cells (19), using a purified CD4 population (16) or using a specific CD4 monoclonal antibody (8). To obtain accurate values that would allow us to fairly compare the results of intracellular cytokine expression between the STM-BFA and STM-MN cell groups, we were required to use the CD8 Ϫ subset rather than the CD4 ϩ subset because of the difficulty encountered in trying to separate CD4 Ϫ cells from CD4 ϩ cells.…”