Changes of Intracellular Signal Pathway of mTOR/S6 in T Cells of Refractory/Relapsed Aplastic Anemia

Objects: To explore

1. the activation state of intracellular signal pathway of mTOR/S6 in refractory/relapsed aplastic anemia (AA);

2. the effect of rapamycin (RAPA) and CTLA-4 immunoglobulin (CTLA-4Ig) on this signal pathway in refractory/relapsed AA.

Methods:

1. Samples were collected from 13 refractory/relapsed AA patients [male: 6, female: 7, media age: 27 years (from 7 to 66years)], 8 newly diagnosed patients with severe aplastic anemia (SAA) [male: 5, female: 3, media age: 21.5 years (from 12 to 58 years)]. The intracellular percentages of p-mTOR, p-S6 and IFN-γ of CD3⁺, CD3⁺CD8⁺ and CD3⁺CD8⁻ T cells in bone marrow were detected by flow cytometry (FCM). 10 iron deficiency anemia (IDA) patients [male: 3, female: 7, media age: 44 years (from 31 to 72 years)] were determined as case controls and normal controls respectively.

2. After exposure to RAPA and CTLA-4Ig respectively, samples were detected by FCM for the expression of p-mTOR, p-S6 and IFN-γ in CD3⁺, CD3⁺CD8⁺ and CD3⁺CD8⁻ T cells in bone marrow, in order to estimate the effect of RAPA and CTLA-4Ig on the pathway of mTOR/S6 in refractory/relapsed AA.

Results:

1. In refractory/relapsed AA, measurement of p-mTOR, p-S6 and IFN-γ in CD3⁺, CD3⁺CD8⁻ and CD3⁺CD8⁺ T cells were (42.42±26.44)%, (44.38±24.95)%, (51.89±27.00)%; (6.47±2.72)%, (9.16±2.89)%, (9.61±5.34)%; (18.87±10.05)%, (13.17±5.88)%, (20.07±15.16)%, respectively; and showed an increased level compared to normal controls [(1.54±1.51)%; P=0.000], [(1.94±1.08)%; P=0.000], [(2.04±2.03)%; P=0.000]; [(0.83±0.82)%; P=0.000], [(0.91±0.88)%; P=0.000], [(0.95±0.93)%; P=0.000]; [(4.42±3.55)%; P=0.000], [(2.35±1.69)%; P=0.000], [(4.73±4.43)%; P=0.004].

2. In newly diagnosed patients with SAA, the levels of p-mTOR and p-S6 in CD3⁺, CD3⁺CD8⁻ and CD3⁺CD8⁺ T cells were (1.71±1.66)%, (2.28±2.15)%, (1.59±1.52)%; (1.23±1.13)%, (1.23±1.07)%, (1.76±1.68)% respectively, and they were similar to normal controls (P>0.05), but significantly lower than those of refractory/relapsed AA (P<0.01). Expression of IFN-γ in CD3⁺, CD3⁺CD8⁻ and CD3⁺CD8⁺ T cells was higher than normal controls with (10.38±3.83)%, (6.11±1.91)%, (13.14±7.05)% (P<0.01). The percentages of CD3⁺IFN-γ⁺ and CD3⁺CD8⁻IFN-γ⁺ were lower than refractory/relapsed AA (P<0.05), while it was comparable for CD3⁺CD8⁺IFN-γ⁺ cells between the two groups (P>0.05).

3. Exposed to RAPA, the expression of p-mTOR, p-S6 and IFN-γ in CD3⁺, CD3⁺CD8⁻ and CD3⁺CD8⁺ T cells decreased markedly (P<0.05) to (12.44±12.41)% (12.60±12.57)%, (16.85±15.64)%; (1.49±1.45)%, (1.46±1.43)%, (1.55±1.54)%; and (4.29±4.23)%, (3.16±3.32)%, (10.70±10.63)% in refractory/relapsed AA.

And treated with CTLA-4Ig could also cause a significant reduction of p-mTOR, p-S6 and IFN-γ in CD3⁺, CD3⁺CD8⁻ and CD3⁺CD8⁺ T cells, which were (6.40±6.13)%, (8.32±7.76)%, (7.18±7.02)%; (1.08±1.08)%, (2.69±2.37)%, (1.60±1.56)%; (1.67±1.60)%, (2.39±2.12)%, (1.30±1.30)%, respectively, (P<0.01).

Conclusions:

1. In refractory/relapsed AA, the signal pathway of mTOR/S6 was activated, and it was quiescent in normal controls and newly diagnosed patients with SAA.

2. The expression of p-mTOR, p-S6 and IFN-γ of this signal pathway in refractory/relapsed AA could be suppressed by RAPA or CTLA-4Ig.

3. The signal pathway of CD28/mTOR/S6/IFN-γ might take part in immune pathogenesis of refractory/relapsed AA, and was sensitive to RAPA and CTLA-4Ig. It was worth exploring the clinical values of the two drugs.