Phenotypic studies of natural killer cell subsets in human transporter associated with antigen processing deficiency

Abstract

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 CD56(bright) 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, (51)Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56(bright) NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56(dim)CD16(-) 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 CD56(bright) 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.

Figure 1. High percentages of CD56^bright NK cells in two symptomatic TAP-deficient patients.

PBMC from four normal donors and the patients were stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry. Expression of CD16 and CD56 is shown on gated CD3⁻CD19⁻ lymphocytes (one representative experiment). Percentages of CD56^bright NK cells among total NK cells are indicated for each donor.

Figure 2. Dramatic over-expression of NKG2A on both CD56^bright and CD56^dim NK cell subsets of symptomatic TAP-deficient patients.

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.

Figure 3. Over-expression of ILT2 on CD56^dim NK cells of symptomatic and asymptomatic patients.

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.

Figure 4. TAP-deficient PHA-induced T cell blasts are resistant to killing by activated autologous NK cells even in the presence of antibodies masking inhibitory receptors.

A standard ⁵¹Chromium release assay was performed with activated NK cells from two normal donors (NK ND A, NK ND B) and of patient EMO (NK EMO) as effectors and TAP-deficient PHA-induced T cell blasts from patient EMO as targets. The effector to target (E/T) ratio was 6/1. Masking Ab were used at a concentration of 10 µg/ml. Indicated values correspond to the percentages of specific lysis.