Phenotypic Studies of Natural Killer Cell Subsets in Human Transporter Associated with Antigen Processing Deficiency

Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56bright cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, 51Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56bright NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56dimCD16− NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56bright NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases

Dual roles for MEF2A and MEF2D during human macrophage terminal differentiation and c-Jun expression.: MEF2A and MEF2D in c-Jun expression during macrophage differentiation

International audienceRecent reports have evidenced a role for the Myocyte Enhancer Factor 2C (MEF2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show here that MEF2A and MEF2D, two other MEF2 family members, are expressed in human primary monocytes and in higher amounts in monocyte-derived macrophages. High levels of MEF2A/MEF2D heterodimers are found in macrophage-differentiated HL60 cells. Chromatin immunopoprecipitations demonstrate that MEF2A is present on the c-Jun promoter, both in undifferentiated and in macrophage-differentiated cells. Moreover, c-Jun expression is derepressed in undifferentiated cells in the presence of Trichostatin A, a histone deacetylase inhibitor, indicating the importance of chromatin acetylation in this process. We show that MEF2A/D dimers strongly interact with HDAC1, and to a lesser extent with HDAC7 in macrophages, whereas low levels of MEF2A/D:HDAC1 complexes are found in undifferentiated cells or in monocytes. Since trichostatin A does not disrupt MEF2A/D:HDAC1 complexes, we analyzed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is upregulated in macrophages. Interestingly, endogenous p300 only associates with MEF2A in differentiated macrophages, indicating that MEF2A/D could activate c-Jun expression in macrophages through a MEF2A/D:p300 activator complex. The targets of MEF2A/D:HDAC1:HDAC7 multimers remain to be identified. Nevertheless, these data highlight for the first time the possible dual roles of MEF2A and MEF2D human macrophages, as activator or as repressor of gene transcription

Antibodies used for flow cytometry

<p>FITC : fluorescein isothiocyanate ; PE : phycoerythrin ; PC5 : phycoerythrin-cyanin 5. *: indirect staining; cells were first stained with the indicated purified Ab, washed, and then stained with a secondary goat anti-mouse Ab conjugated to PE. All other Ab were used in directly fluorochrome-conjugated forms.</p

NK cell subsets in TAP-deficient patients and healthy control donors

<p>Values correspond to the cells in the different subsets expressed as percentages of total peripheral blood NK cells. HD: healthy donor; EHA: father of patients EFA and EMO; EFA, EMO: symptomatic TAP2-deficient patients (siblings); SFH, DFH: asymptomatic TAP2-deficient patients (siblings).</p

Dramatic over-expression of NKG2A on both CD56^bright and CD56^dim NK cell subsets of symptomatic TAP-deficient patients.

<p>Mean fluorescence intensities (MFI) of NKG2A were expressed relative to the values of a randomly chosen healthy donor arbitrarily considered as 100%. Mean values of different subject groups were calculated. ND: normal donors (n = 17); CF: cystic fibrosis patients (N = 2), V: vasculitis patients (n = 5), inf. R.I.: infected respiratory insufficiency (n = 5), uninf. R.I.: uninfected respiratory insufficiency (n = 5); EFA, EMO: symptomatic TAP-deficient patients; DFH, SFH: asymptomatic TAP-deficient patients.</p

NK cell subsets in additional healthy donors and patients with diseases other than TAP deficiency

<p>HD 8–HD 17: healthy donors (different from those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001033#pone-0001033-t002" target="_blank">table 2</a>); 1–17: patients 1–17 with diseases other than TAP deficiency (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001033#pone-0001033-t003" target="_blank">table 3</a> for details).</p

Over-expression of ILT2 on CD56^dim NK cells of symptomatic and asymptomatic patients.

<p>Mean fluorescence intensities (MFI) of ILT2 were expressed relative to the values of a randomly chosen control donor arbitrarily considered as 100%. Mean values of different subject groups were calculated. ND: normal donors (n = 17); CF: cystic fibrosis patients (N = 2), V: vasculitis patients (n = 5), inf. R.I.: infected respiratory insufficiency (n = 5), uninf. R.I.: uninfected respiratory insufficiency (n = 5); EFA, EMO: symptomatic TAP-deficient patients; DFH, SFH: asymptomatic TAP-deficient patients.</p