Duck Cho

Primary Anti-D Alloimmunization Induced by "Asian Type" RHD (c.1227G>A) DEL Red Cell Transfusion

The Rh blood group system is the second most clinically significant blood group system after the ABO blood group system [1]. Extremely weak D variants known as DEL, which are weaker than Du, are hardly detectable by basic serologic typing except absorption-elution techniques and frequently require molecular studies for confirmation [2]. According to the previous review, the 678 DELs found by elution or DNA detection were genotyped, and more than 98% (667/678) presented the RHD (c.1227G>A) allele, which predominates in East Asians [3]. Thus, it is known as the Asian type. Here, we present a case of primary anti-D alloimmunization induced by the transfusion of packed red blood cells (RBCs) from two Korean donors, both of whom coincidentally had DEL phenotypes. n nA 64-yr-old Caucasian male was admitted to our hospital for surgery for prostate adenocarcinoma. His blood type was group A, D-negative. The patient had no history of transfusion. Two days after admission, two units of crossmatch-compatible blood group A, D-negative packed RBCs from two separate donors were administered. The result of a pre-transfusion antibody screening test (BioVue, Ortho Clinical Diagnostics, Raritan, NJ, USA), an indirect antiglobulin test with column agglutination, was negative. No initial adverse effect of transfusion was observed. At day 12, the antibody screening test became positive and anti-D was identified in the recipients serum (Resolve Panel A, Ortho Clinical Diagnostics). An autocontrol and a direct antiglobulin test (DAT) showed no visible agglutination. n nAnti-D development after transfusion in this patient was unexplainable, and possible analytical errors were ruled out. An antibody screening test performed by using previously taken blood samples yielded strongly positive results (4+) on days 7, 9, and 12 and a negative result on day 5. This implied that anti-D developed between day 5 and day 7. n nThe remaining pre-sealed portions of two transfused RBC units were sent to the Korean Rare Blood Program Reference Laboratory (Seoul National University Bundang Hospital) for confirmation of the RHD variants. The RHD genotype was analyzed according to the previously described methods [4]. Surprisingly, the RhD genotype results for both donors were RHD (c.1227G>A). They presented the Ccee and CCEe phenotypes (Table 1). The two RHD (c.1227G>A)-positive RBC units were transfused at day 2 after admission, and the above observations suggest that anti-D alloimmunization caused anti-D to be detected more than 3 days later. n n n nTable 1 n nBlood group immunogenetic results of two donors n n n nDEL can cause anti-D alloimmunization despite small amounts of D antigens on RBCs. Several cases of anti-D alloimmunization caused by transfusion from DEL donors have been reported [4,5,6] (Table 2). Although 16% of serologically D-negative Korean blood donors were known to be DEL, only one patient of anti-D alloimmunization has been reported in Korea [4]. In our case, the patient received serologically D-negative RBCs from two donors, who were later shown to have the DEL phenotype by RHD genotyping. Two separate serologically D-negative RBC units with a DEL phenotype may not be a coincidence. In Germany and Upper Austria, RHD genotyping of D-negative donors is routinely performed as a screening method at first-time donation [7,8]. The prevalence of RHD gene carriers was 0.21% for six years, and approximately a half of them had a DEL phenotype, according to the data from Germany [7]. In Upper Austria, of 23,330 serologically D-negative samples, 94 showed one or more RHD markers from among 20 RHD markers located in exons 4, 7, and 10 [8]. However, according to the national transfusion guidelines from South Korea, serologically D-negative units are not tested for true D-negative and DEL phenotypes. Therefore, it is possible for DEL-type packed RBC units to be transfused to D-negative recipients. If RHD PCR is adopted as an initial screening method for D-negative blood, the number of cases with DEL phenotypes being serologically mistyped as general D-negative will decrease. However, it would increase costs and require additional time. n n n nTable 2 n nCharacteristics of anti-D immunization by the DEL RBCs in literature n n n nPrevious investigations reported that the presence of RHD genes was strongly related to RhC phenotype, with a high frequency of RhC(+) in serologically RhD-negative blood [9,10]. In an earlier study of D-negative Koreans, all except one (97.6%) of 42 Asian type (c.1227G>A) individuals showed a RhC phenotype [9]. Our study also showed the RhC(+) phenotype in both donors, who have Ccee and CCEe phenotypes. Thus, we agree with Wang et al. [10] that a laboratory protocol for blood banks that includes RhC phenotyping and confirmatory RHD PCR would be helpful for detecting DEL RBCs. Additional nationwide Korean data concerning the incidence of the RhC(+) phenotype in DEL individuals needs to be collected.

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor

BACKGROUND AIMSnFew studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells.nnnMETHODSnThe expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96u2009h after irradiation of breast cancer cell lines for migration and blocking assays.nnnRESULTSnThe activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R2u2009=u20090.91; Pu2009<0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (Pu2009=u20090.043) and SKBR3 (Pu2009=u20090.043) cells, but not in MDA-MB231 (Pu2009=u20090.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines.nnnCONCLUSIONSnOur data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer.

An effective diagnostic strategy for accurate detection of RhD variants including Asian DEL type in apparently RhD-negative blood donors in Korea.

The purpose of this study was to provide an effective RHD genotyping strategy for the East Asian blood donors.

Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: Reference range, influence of age and sex, and correlation between NK cell receptors and cytotoxicity

BACKGROUNDnThe purpose of this study was to identify CD56bright and CD56dim natural killer (NK) cell subsets and analyze their receptors expression in a healthy Korean population, and to determine whether receptor expression correlates with age, sex, and cytotoxicity.nnnMATERIALS AND METHODSnWe performed multicolor flow cytometry assays to analyze the expression of various NK cell receptors (CD16, NKG2A, NKG2C, NKG2D, CD57, DNAM-1, CD8a, CD62L, NKp30, and NKp46) on both CD3-/CD56dim and CD3-/CD56bright NK cells in whole-blood samples from 122 healthy donors. The expression of these receptors was compared according to age (<30years, n=22, 30-60years, n=73 and >60years, n=27) and gender (male, n=61, female, n=61). NK cell cytotoxicity assays were performed with peripheral blood mononuclear cells (PBMCs) from 18 individuals. The results were compared to the expression levels of NKp30 and NKp46 receptors.nnnRESULTSnA normal reference range for NK cell receptor expression in two NK cell subsets was established. NKp46 and NKG2D expression gradually decreased with age (p<0.01 and p<0.05, respectively) whereas NK cell proportion and numbers, frequencies of CD56dim cells, and CD57 expression increased with age (p<0.01 in all cases). Men showed greater NK cell proportion and numbers, frequencies of CD56dim cells, and CD57 expression compared to those of women (p<0.05 and p<0.001; p<0.01 and p<0.01, respectively). Notably, the expression of NKp46 was negatively correlated with NK cell frequency (r=-0.42, p<0.001). Furthermore, NK cell cytotoxicity was found to positively correlate with NCR expression (p=0.02), but not NK cell proportion (p=0.80).nnnCONCLUSIONnWe have established a profile of NK cell surface receptors for a Korean population, and revealed that age and gender have an effect on the expression of NK cell receptors in the population. Our data might explain why neither NK cell numbers nor proportions correlate with NK cell cytotoxicity.

Human U87 glioblastoma cells with stemness features display enhanced sensitivity to natural killer cell cytotoxicity through altered expression of NKG2D ligand

BackgroundGlioblastoma (GBM) is one of the most lethal tumors with a poor prognosis. Its inevitable recurrence is frequently explained by the presence of cancer stem cells. We aimed to show that human GBM cells with stemness features are more sensitive to natural killer (NK) cells than GBM cells without stemness characteristics.MethodsNatural killer cell cytotoxicity was measured using flow cytometry in neurosphere-forming U87 GBM cells cultured with neurobasal media (NBE condition) and compared with that in serum-cultured U87 GBM cells (serum condition). Cytotoxicity was examined after addition of blocking NKG2D monoclonal antibodies. The expression profile of NK ligands of NK cells were investigated by reverse transcription polymerase chain reaction and western blot analysis in the U87 GBM cells in both conditions.ResultsNBE U87 cells showed higher cytotoxicity to NK cells than serum U87 cells did (55 vs 35% at an effector to target cell ratio of 5:1). The increased cytotoxicity was diminished in NBE U87 cells by a larger gap than in serum U87 cells by adding NKG2D blocking antibodies. Of the NKG2D ligands, the expression of ULBP1 and ULBP3 was relatively increased in NBE U87 cells compared to serum U87 cells.ConclusionsU87 GBM cells with stemness features demonstrate increased cytotoxicity to NK cells in association with altered NKG2D ligand expression of NK cell activating receptor. Applying immune modulation to GBM treatment may be a promising adjuvant therapy in patients with intractable GBM.

Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells

Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCRαβ(-)TCRγδ(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-γ. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells.

A novel cis-AB variant allele arising from a de novo nucleotide substitution c.796A>G (p.M266V) in the B glycosyltransferase gene.

Cis‐AB, a rare ABO variant, is the result of a mutated ABO gene that produces a glycosyltransferase enzyme with dual A and B glycosyltransferase activity. It may lead to ABO discrepancies and a delay in establishing the blood group. To date, there have been no reports of a de novo mutation leading to a cis‐AB allele.

Cellular immunotherapy as a beacon of hope for hematological malignancies.

Recently, successful cancer immunotherapy has aroused great interest in application of this approach in hematological malignancies. Immune responsiveness is a key clinical feature of hematological malignancies. Indeed, the efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) largely derives from graft-versus-tumor effects that highlight the ability of the immune system to specifically and effectively eliminate tumors [1]. Here, we describe the status of cellular immunotherapy in hematological malignancies and discuss its future perspectives.

Effect of irradiation-induced intercellular adhesion molecule-1 expression on natural killer cell-mediated cytotoxicity toward human cancer cells

BACKGROUND AIMSnIrradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell-mediated cytotoxicity.nnnMETHODSnExpression levels of ICAM-1 on the target cell surface before and after irradiation of six human cancer cell lines (HL60, SKBR-3, T47D, HCT-116, U937 and U251) were analyzed by flow cytometry. Ex vivo expansion of NK cells from human peripheral blood mononuclear cells was performed by co-culture with irradiated K562 cells. The related adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) on NK cells was analyzed by flow cytometry. An enzyme-linked immunosorbent assay was used to detect interferon-γ (IFN-γ), and WST-8 assays were performed to check NK cell cytotoxicity. Finally, blocking assays were performed using monoclonal antibodies against ICAM-1 or LFA-1.nnnRESULTSnLFA-1 expression increased on NK cells after expansion (Pu2009<0.001). The expression of ICAM-1 was significantly upregulated by irradiation after 24u2009h in various cell lines, including HL60 (Pu2009<0.001), SKBR-3 (Pu2009<0.001), T47D (Pu2009<0.001) and U937 (Pu2009<0.001), although the level of expression depended on the cell line. ICAM-1 expression was extremely low before and after irradiation in U251 cells. NK cell-mediated cytotoxicity increased after irradiation of HL60 (Pu2009<0.001), SKBR-3 (Pu2009<0.001), T47D (Pu2009=u20090.003), and U937 (Pu2009=u20090.004) cells, in which ICAM-1 expression was significantly increased after irradiation. IFN-γ production by NK cells in response to HL60 (Pu2009<0.001) and T47D (Pu2009=u20090.011) cells significantly increased after irradiation. NK cell-mediated cytotoxicity against irradiated SKBR-3 (Pu2009<0.001) and irradiated T47D cells (Pu2009=u20090.035) significantly decreased after blocking of ICAM-1. Blocking of LFA-1 on NK cells resulted in reduced cytotoxicity against irradiated HL60 (Pu2009<0.001) and irradiated SKBR-3 (Pu2009<0.001).nnnCONCLUSIONSnIrradiation upregulates ICAM-1 expression on the surface of human cancer cells and enhances activated NK cell-mediated cytotoxicity. Therefore, irradiation combined with NK cell therapy may improve the antitumor effects of NK cells.

Short-Term Outcomes of ABO-Incompatible Living Donor Kidney Transplantation With Uniform Protocol: Significance of Baseline Anti-ABO Titer

Antibody-mediated rejection (AMR) is one of the major causes of poor outcomes in ABO-incompatible kidney transplantation (ABOi KT). Studies investigating AMR risk factors found that anti-ABO titer is a major issue. However, the significance of antibody titer has been debated. This retrospective study analyzed AMR risk factors in 59 patients who underwent ABOi KT between August 2010 and January 2015. We also analyzed AMR risk factors in recipients with high anti-ABO baseline titers (≥1:64 on dithiothreitol at 37°C phase orxa0≥1:256 on antihuman globulin phase). The 2-year patient survival rate was 95.8%, and the 2-year graft survival rate was 94.9%. Nine patients (15.3%) experienced clinical (6xa0of 59 [10.2%]) or subclinical (3 of 59 [5.1%]) AMR. One patient experienced graft loss from hyperacute rejection. AMR risk factor analysis revealed that baseline antibody titer was associated with incidence of AMR. In patients with high baseline titers, low doses of rituximab (200-mg single-dose), an antibody against CD20, was predictive for AMR. Six patients who received pretransplant intravenous immunoglobulin did not experience AMR even when they had high baseline antibody titers. Our results indicate that a high baseline antibody titer affected the incidence of AMR. ABOi KT candidates with high baseline titers need to undergo an intensified preconditioning protocol, including high-dose rituximab (375 mg/m(2)) and intravenous immunoglobulin.