Quantities and Functions of NK Cells in Severe Aplastic Anemia

Introduction

Severe aplastic anemia (SAA) is an autoimmune disease characterized by bone marrow failure. Natural killer (NK) cells are a key component in the innate immunity and adaptive immune. Our team have previously investigated the quantity and primary functions of NK in SAA. Here, we tend to present the extensive study on the roles NK has played in SAA.

Methods

We successively collected patients with untreated SAA, SAA response to immunosuppressive therapy (SAA-CR+PR), and healthy controls (HC) as research participants. Quantities of circulating NK and their subsets (CD56^bright NK and CD56^dim NK) were identified, the expressions of surface receptors,intracelluar protein and intracellular cytokines were analyzed by flow cytometry (FCM). Plasmatic IL-2 and IL-12 levels were measured by ELISA. NK cells were cultured with K562 cells. The apoptotic rate of K562 was measured to detect killing functions of NK. NK cells were co-cultured with CD4⁺ T , CD8⁺ T and myeloid Dendritic Cells (mDCs) from SAA. Quantities of Th1, Th2, Th17 and Treg, expressions of perforin and Granzyme B in CD8⁺ T cells and expressions of CD80 and CD86 on mDCs were analyzed by FCM. The mRNA expressions of T-bet, GATA3, RORγt and FOXP3 were measured by qRT-PCR. Proliferation rates of CD8⁺ T cells and mDC were detected by CCK-8. PeripheralNK cells were sorted and cultured with rhIL-2 and rhIL-12. The supernatants were detected for the cytokines produced by NK. The cytokines and their upstream signal molecules were analyzed by qRT-PCR and Western Blot. Finally, we investigated the quantities and the surface receptors of NK using the mouse model of lymphocyte infusion-induced bone marrow failure to confirm their functions.

Results

The percentages of NK/lymphocytes and CD56^brightNK/NK in SAA were significantly lower than in HC. The absolute number of NK cells in SAA was positively correlated with PLT, absolute number of Reticular cells (Ret) and percentage of Ret (p <0.01). NK cells in SAA expressed higher levels of NKp46, CD158a and perforin compared with those in HC. The percentage of DNAM-1⁺ NK was higher than that in HC (p=0.007). After PMA stimulation, NK cells in SAA produced lower level of IFN-γ and higher level of IL-10 and TGF-β compared with those in HC. The plasmatic levels of IL-2 and IL-12 in SAA were higher than in SAA-CR+PR and HC. The apoptotic rate of K562 cells when co-cultured with NK cells (1:1) was significantly higher than in HC.IL-2stimulatedNK cells from SAA expressed higher percentage of CD107a⁺ than from HC (p=0.017) when co-cultured with autologous activated CD4⁺T cells. After co-cultured with NK cells from SAA, the ratio of Th1/Th2 and Th17/Treg was decreased. The expressions of perforin and Granzyme B in CD8⁺ T cells were higher when co-cultured with NK from HC than in non-coculture group. The cell inhibition rate of mDCs when co-cultured with NK from SAA was higher than from HC. The expression of CD80 on mDCs when co-cultured with NK from HC was up-regulated.NK cells from SAA secreted lower level of IFN-γ, expressed lower levels of IFN-γ and STAT4 mRNA and phosphorylated STAT4. NK cells from SAA secreted higher levels of IL-10 and TGF-β, expressed higher levels of IL-10, TGF-β and p38 mRNA and phosphorylated p38. Bone marrow failure mouse model showed the quantities of NK cells weredecreased, the expression of NKG2A on NK was elevated. After the treatment of Cyclosporin A (CsA), the quantities NK cells recovered, the expression of NKG2D was elevated while NKG2A was not changed.

Conclusions

Insufficient amount of NK cells was shown in SAA patients. The lower quantity of NK cells the patients had,the more severe the disease was. NK cells in SAA were functionally activated and exhibited strong killing abilities. NK cells in SAA produced less Type Ⅰ cytokines and more Type Ⅱ cytokines. Plasmatic levels of IL-2 and IL-12 were elevated in SAA. NK cells from SAA mediated in the differentiation of CD4⁺ T cells by influencing the balance of Th1/Th2 and Th17/Treg. NK cells from SAA might be able to down-regulate the cytotoxicity activity of CD8⁺ T cells and the expressions of co-stimulatory molecules on mDCs. The cell signal pathway JAK-STAT4-IFN-γ in NK cells from SAA was down-regulated while pathway p38 MAPK-IL10/TGF-β was up-regulated. Bone marrow failure mouse model suggested the impairment of both quantities and activating status of NK.

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