ALBERT

Description: The fine phase transformation process of chalcocite (Cu2S) leaching in acidic ferric sulfate solution was studied by leaching experiments and synchrotron radiation X-ray diffraction (SRXRD) tests. The results showed that the dissolution process of chalcocite was divided into two stages. In the first stage, Cu2S was firstly transformed to Cu5FeS4 and Cu2−xS, then the galvanic effect between Cu5FeS4 and Cu2−xS accelerated the dissolution process of Cu1.8S → Cu1.6S → CuS, and finally Cu5FeS4 was also transformed to CuS. While in the second stage, CuS was transformed to elemental sulfur, which formed the passivation layer and inhibited the leaching of chalcocite. Specifically, Cu5FeS4 was detected during the chalcocite leaching process by SRXRD for the first time. This research is helpful for revealing the detailed leaching process of chalcocite.

Description: Background:Educated NK cells prevent autoreactive behavior but also permit cytotoxicity against target cells that have down-regulated HLA class I expression. When and how the process of education occurs has not been clearly discerned. Several groups reported that both the donor and host MHC could influence NK cell education in mouse models. In humans, Dulphy et al demonstrated that NK-cell education is shaped by donor HLA genotype. Moreover, our previous study found NK-cell education was shaped by host HLA genotype post allo-HSCT. However, due to the lack of single-KIR+ NK cells, functional analysis limited the full evaluation of the interaction between donor/host HLA and donor inhibitory KIR, so the contribution of donor HLA could not be excluded. Aims: In this research, we have investigated the relative contributions of donor and recipient HLA to NK cells education, the interplay between functional reconstitution and the involvement of donor/host HLA interaction in NK cell control of leukemia cells. Methods: Two cohorts of patients were enrolled in this study. We first prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore NK cell phenotypes and functional reconstitution. From June 2012 to April 2016, 276 AML/MDS patients that underwent haploidentical transplantation were enrolled in the second cohort to analyze the effect of donor-host KIR-HLA combinations on relapse post transplantation. Molecular HLA typing and KIR genotyping were performed according to the manufacturer's instructions (One Lambda, Canoga Park, CA, USA). Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry. The cytotoxicity and cytokine secretion of NK cells was determined using CD107a expression and IFN-γ production against the K562 cell line. Single-KIR+ NK cells were grouped into the following groups: (A) nsKIR: where both hosts and donors lacked HLA ligands for one donor KIR; (B) d-rsKIR, where donors and hosts, encoded HLA ligands for donor KIRs; (C) dsKIR, where donors, but not hosts, encoded HLA ligands for donor KIR; and (D) rsKIR, where hosts, but not donors, encoded HLA ligands for donor KIR. Results: 1. Donor KIR ligated by both donor and host HLA is associated with better single-KIR+ NK cell education among the same patients. KIR2DL2/L3 single+ NK cell exhibited higher reactivity compared to KIR2DL1 single+ NK cell in pairs of donors C1C1 or C1C2 and host C1C1. KIR2DL2/L3 single+ NK cell exhibited higher reactivity than KIR3DL1 single+ NK cell in pairs of donors Bw4C1Cx and host C1Cx. KIR2DL1 single+ NK cell exhibited comparable reactivity with KIR2DL2/L3 single+ NK cell in pairs of donor Bw4C1C2 and host Bw4C1C2. 2. Donor KIR ligated by both donor and host HLA contribute to better single-KIR+ NK cell education among the same single-KIR+ NK cells. KIR2DL2/L3 single+ NK cell in the group of d-rsKIR (C1Cx-C1Cx) exhibit higher reactivity compared with other groups (dsKIR (C1Cx-C2C2), rsKIR (C2C2- C1Cx)). KIR2DL1 single+ NK cells in group of d-rsKIR (C2Cx-C2Cx) exhibited higher reactivity compared with other groups (nsKIR (C1C1-C1C1), dsKIR (C2Cx-C1C1), rsKIR (C1C1-C2Cx)). KIR3DL1 single+ NK cells in groups of d-rsKIR (Bw4Bwx-Bw4Bwx) exhibited higher reactivity compared with other groups (nsKIR (Bw6Bw6-Bw6Bw6), dsKIR (Bw4Bwx-Bw6Bw6), rsKIR (Bw6Bw6-Bw4Bwx)). 3. Both donor and host HLA must coexist for maximum education of NK cells given donor 3 inhibitory KIRs. When both of donor and host presenting all HLA (Bw4C1C2), we showed a remarkable hierarchy of responses among NK populations. NK cells with two inhibitory KIRs for self-HLA exhibited higher NK responsiveness (CD107α and IFN-γ) compared with single KIR+ NK cells. NK cells with 3 inhibitory KIRs for self-HLA exhibited maximum responsiveness. 4. Both donor and host exhibiting all HLA (Bw4C1C2) for donor 3 inhibitory KIRs contributes to least relapse following haploidentical allo-HSCT. In the second cohort, the lowest relapse rate was found in d-rsKIR group (n=31, 0%) compared with rsKIR group (n=55 ,0% vs. 10.0±4.9%, P=0.115), dsKIR group (n=33, 0% vs 14.9%±7.0%, P=0.039), or nsKIR group (n=156, 0% vs. 18%±3.5%, P=0.022). Summary: This study demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients. Disclosures No relevant conflicts of interest to declare.

Description: Background: DNAM-1 (DNAX accessory molecule-1, CD226) is a costimulatory molecule that is constitutively expressed by NK cells and CD8+ T cells. Interaction between DNAM-1 on NK cells and CD8+ T cells with its ligands on target cells triggers cell-mediated cytotoxicity and cytokine production. Previous mouse model had showed that the expression of DNAM-1 is associated with NK education. Moreover, Monika's group found that DNAM-1 serves as an intrinsic, readout-independent marker for NK cell education in health donor. Our previous study had demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients post allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the roles of DNAM-1 in NK education post allo-HSCT were unknown. Aims: In this study, we have investigated the contribution of DNAM-1 expression to NK education post transplantation. Methods :We prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore the NK cell dynamic education at days 30, 90 and 180 post-transplant. Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry to evaluate of the phenotype as well as functional recovery of NK cells with different maturation expression levels of NKG2A, KIR, and CD57, as well as of activating receptors (NKp30, NKp46, NKG2D, DNAM-1) and CD25, CD122 as well as CD107a and IFN-gamma on NK cells. To study the correlation between the expression of DNAM-1 and effect of donor and host HLA on NK cell education, the expression of DNAM-1 on single-KIR+ NK cells (exhibiting NKG2A, KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) was compared based on the combination between donor/host HLA and donor inhibitory KIR. Results: The DNAM-1 expression on single-KIR+ NK cell ligated by sole donor HLA, or sole host HLA, or both donor and host HLA is higher compared to those single-KIR+ NK cells lacking ligands from donor or host or both. From the donor point of view, when donor only presented C1C1 ligand for KIR2DL2/L3, the MFI of DNAM-1 on single KIR2DL2/L3+ NK cells was significantly higher than KIR2DL1+ NK cells and KIR3DL1+ NK cells at 90day (P

Description: Backgroud: Refractory cytomegalovirus (CMV) infection remains important causes of morbidity and mortality after allogeneic hematopoietic stem cells transplantation (allo-HSCT). Previous researches reported that adaptive immunity, such as CD8+ CMV-CTL, plays an important role in the in the control of refractory CMV infection. In mouse, Lanier et al. found there existed subsets of adaptive NK cells with the features of expanding, contracting after control of mouse CMV, and generating long-lived "memory" NK cells. In human, these adaptive NK cells were initially identified based on the high expression of the NKG2C which against HCMV through their cytotoxic potential and the production of TNF-α and IFN-γ upon Ab-mediated stimuli in vitro. Meanwhile, the expression levels of the NKG2C+ adaptive NK cells has been positively correlated with the NKG2C copy number. Several researchers had found that NKG2C+ adaptive NK cells persistent expanded and were potent producers of IFN-γ during CMV reactivation after solid-organ transplant or allo-HSCT. However, the role of NKG2C+ adaptive NK cells on refractory CMV reactivation were still unknown. Whether the rapid reconstitution of NKG2C+ adaptive NK cells can reduce the refractory CMV reactivation merit to be investigated. Aims:In this research, we had investigated the impacts of the quantity and quality recovery of NKG2C+ adaptive NK cells on the occurrence of refractory CMV infection. Method: At first, continuous 364 patients underwent allo-HSCT since June 2012 to February 2016 were prospectively enrolled and we retrospectively analyzed the correlationship between their donor NKG2C genotype and refractory CMV infection occurrence post transplantation. Secondly, the second cohort comprising continuous 125 patients underwent allo-HSCT since May 2016 to April 2017 were prospectively enrolled to analyzed the effect of donor NKG2C genotype on NKG2C+ adaptive NK cell recovery as well as the effect of NKG2C+ adaptive NK cell recovery on refractory CMV infection. The cytotoxicity of reconstituting NKG2C+ adaptive NK cells were evaluated against K562 cells, AD169 CMV stain infected MRC-5 cells, and UL40 peptide pulsed 721.221 cells to detect the anti-tumor or anti-CMV function of NKG2C+ adaptive NK cells. Results: Firstly, from the first cohort, we found that donor NKG2C gene deletion was an independent prognostic factor for refractory CMV reactivation (P=0.010) through the multivariate analysis. Then, through in-depth investigation from the second cohort, we found that the absolute cell counts recovery and anti-tumor function of NKG2C+ adaptive NK cells were both significantly lower in patients accepting NKG2C+/del donor than those patients accepting NKG2C+/+ donors at day 30, 90, and180, respectively after transplantation. There was no NKG2C+ adaptive NK cell recovery post transplantation in the patients who accepted NKG2Cdel/del donors. Meanwhile, anti-CMV function recovery of NKG2C+ adaptive NK cells in patients with NKG2C+/del donors were significantly lower than those patients with NKG2C+/+ donors at day 30 post transplantation. Furthermore, we further analyzed the relationship between the early reconstitution of NKG2C+ adaptive NK cells and refractory CMV infection occurrence. The patients were divided into three groups: no CMV, CMV reactivation (persistent time of CMV infection 2 weeks). We found that the absolute cell counts of NKG2C+ adaptive NK cells in refractory CMV group was significantly lower than that of other two groups at day 30 transplantation. When the patients were devided into high and low level groups based on the ROC cut-off percentage of NKG2C+ adaptive NK cells (1.42%), the result revealed that the patients with lower level of NKG2C+ adaptive NK cells at day 30 post-HSCT had an higher cumulative incidence rate of refractory CMV infection (81.1%) comparing with the higher one (40.5%) (P=0.0014). Moreover, Cox regression model further demonstrated that the lower level of NKG2C+ adaptive NK cells at day 30 post-HSCT was significantly associated with refractory CMV infection (HR=2.578, 95% CI 1.379-4.21, P=0.003). Summary/Conclusion: Our results indicated that donor NKG2C deletion damaged the reconstitution of NKG2C+ adaptive NK cells after allo-HSCT, therefore increased the occurrence of refractory CMV infection post transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment strategy for hematological malignancies. However, graft-versus-host disease (GVHD) is a common complication after allo-HSCT resulting from the activation, amplification and secretion of numerous inflammatory factors related to donor alloreactive T cells that damage host tissues and organs, mainly in the gastrointestinal tract, liver and skin. Notably, natural killer (NK) cells represent the first donor-derived lymphocyte population to recover after allo-HSCT and generally are observed within the first month after allo-HSCT. NK cells express a series of immune receptors that identify relevant ligands on target cells and maintain the immune balance between NK cell activation and tolerance. Previous studies have shown that the number of NKG2A+ cells is decreased in patients with chronic GHVD after HSCT. However, the relationship between NKG2A+ NK cells and aGVHD has not been characterized. In addition, the role of NKG2A+ cells in aGVHD disease progression and the mechanism underlying NKG2A+ cell immunoregulation have not been clearly explained. Objective: In this study, we used peripheral blood from GVHD, non-GVHD paired specimens and healthy donors to address the underlying mechanism by which NKG2A+ NK cells regulate T cells after HSCT. Methods: We detected the specimens using flow cytometry from two independent cohorts, which were prospective cohort and paired cohort. Futher, we performed experiments in vitro to investigate the potential role of NKG2A+ NK cells on T cells. Results: Here, we found that, compared with non-GVHD subset, NKG2A+ NK cells percentage and absolute cell counts were significantly reduced in GVHD patients after HSCT. Moreover, the reduction of NKG2A+ NK cells in GVHD patients was ascribed to its increased apoptosis and decreased proliferation capacity, while retaining a strong graft-versus-leukemia (GVL) effect. In vitro assay showed that when co-cultured T cells with NKG2A+ NK cells, the T cells secreted IFN-r level was significantly reduced, while the IL-4 level was increased. Moreover, CD25 expression level was decreased, while the CD4+CD25+FOXP3+ cells number was increased. In addition, the NKG2A+ NK cells induced T cell apoptosis and decreased T cell proliferation during the coculture process. Significantly, NKG2A+ mainly regulated activated but not resting T cells. In vivo assay showed that serological IL-10 level in GVHD subset was evidently lower than those of non-GVHD subgroup, the IL-1β, IFN-r and TNF-a level was however higher in the GVHD subgroup. Furthermore, the percentage of NKG2A+ NK cells from GVHD patients was markedly increased by the presence of exogenous IL-10, but not by other cytokines. However, this phenotype was not observed at non-GVHD patients. Together, the GVHD might be ascribed to lower IL-10 induced NKG2A+ NK cells reduction, which further overactivate T cells after HSCT. Discussion and Conclusion: Overall, we herein observed reduced proportions and absolute cell counts of NK cells and NKG2A+ subsets in patients with acute GVHD after allo-HSCT. The causative association between NK cell numbers, NKG2A+ subsets and GVHD remains debatable. Based on our results, speculating that reduced proportions of NKG2A+ subsets in patients after allo-HSCT are associated with acute GVHD due to their interplay with the patient's donor-derived alloreactive T cells is tempting. Disclosures No relevant conflicts of interest to declare.