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Background aims. Natural killer (NK) cell-based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. T o achieve this, optimization of protocols is required. Methods. Mature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34+ hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay. Results. Addition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90-31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell--derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors. Conclusions. Mature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy. [ABSTRACT FROM AUTHOR]

Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D 2 nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N = 3276) but not in recipients of a living-donor kidney (N = 1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32-2.53; P = .0003). These antibodies occur independently from donor-specific anti-HLA antibodies (DSA) or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation., (© 2019 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Few studies have evaluated the effect of different immunosuppressive strategies on long-term kidney transplant outcomes. Moreover, as they were usually based on historical data, it was not possible to account for the presence of pretransplant donor-specific human-leukocyte antigen antibodies (DSA), a currently recognized risk marker for impaired graft survival. The aim of this study was to evaluate to what extent frequently used initial immunosuppressive therapies increase graft survival in immunological low-risk patients., Methods: We performed an analysis on the PROCARE cohort, a Dutch multicentre study including all transplantations performed in the Netherlands between 1995 and 2005 with available pretransplant serum (n = 4724). All sera were assessed for the presence of DSA by a luminex single-antigen bead assay. Patients with a previous kidney transplantation, pretransplant DSA or receiving induction therapy were excluded from the analysis., Results: Three regimes were used in over 200 patients: cyclosporine (CsA)/prednisolone (Pred) (n = 542), CsA/mycophenolate mofetil (MMF)/Pred (n = 857) and tacrolimus (TAC)/MMF/Pred (n = 811). Covariate-adjusted analysis revealed no significant differences in 10-year death-censored graft survival between patients on TAC/MMF/Pred therapy (79%) compared with patients on CsA/MMF/Pred (82%, P = 0.88) or CsA/Pred (79%, P = 0.21). However, 1-year rejection-free survival censored for death and failure unrelated to rejection was significantly higher for TAC/MMF/Pred (81%) when compared with CsA/MMF/Pred (67%, P < 0.0001) and CsA/Pred (64%, P < 0.0001)., Conclusion: These results suggest that in immunological low-risk patients excellent long-term kidney graft survival can be achieved irrespective of the type of initial immunosuppressive therapy (CsA or TAC; with or without MMF), despite differences in 1-year rejection-free survival., (© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: Pre-transplant donor-specific anti-human leucocyte antigen (HLA) antibodies (DSAs) are associated with impaired kidney graft survival while the clinical relevance of non-donor-specific anti-HLA antibodies (nDSAs) is more controversial. The aim of the present paired kidney graft study was to compare the clinical relevance of DSAs and nDSAs., Methods: To eliminate donor and era-dependent factors, a post hoc paired kidney graft analysis was performed as part of a Dutch multicentre study evaluating all transplantations between 1995 and 2005 with available pre-transplant serum samples. Anti-HLA antibodies were detected with a Luminex single-antigen bead assay., Results: Among 3237 deceased donor transplantations, we identified 115 recipient pairs receiving a kidney from the same donor with one recipient being DSA positive and the other without anti-HLA antibodies. Patients with pre-transplant DSAs had a significantly lower 10-year death-censored graft survival (55% versus 82%, P=0.0001). We identified 192 pairs with one recipient as nDSA positive (against Class I and/or II) and the other without anti-HLA antibodies. For the patients with nDSAs against either Class I or II, graft survival did not significantly differ compared with patients without anti-HLA antibodies (74% versus 77%, P = 0.79). Only in patients with both nDSAs Class I and II was there a trend towards a lower graft survival (58%, P = 0.06). Lastly, in a small group of 42 recipient pairs, 10-year graft survival in recipients with DSAs was 49% compared with 68% in recipients with nDSAs (P=0.11)., Conclusion: This paired kidney analysis confirms that the presence of pre-transplant DSAs in deceased donor transplantations is a risk marker for graft loss, whereas nDSAs in general are not associated with a lower graft survival. Subgroup analysis indicated that only in broadly sensitized patients with nDSAs against Class I and II, nDSAs may be a risk marker for graft loss in the long term., (© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients. Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay. Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P =0.93). Patients without DSA had a 10-year graft survival of 78%. Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure., (Copyright © 2018 by the American Society of Nephrology.)

Between 2001 and 2012, the number of unrelated donors registered worldwide increased from 7 to 21 million, and the number of public cord blood units increased to over 500,000. We addressed the question of whether this expansion resulted in higher percentages of patients reaching transplantation. Unrelated donor searches were evaluated for 3,124 eligible patients in the Netherlands in two cohorts (2001-2006, n=995; 2007-2012, n=2129), comparing results for patients of Northwestern European and non-Northwestern European origin. Endpoints were 'donor found' and 'transplantation reached'. The substantial growth of the donor inventory over the period studied did not increase the median number of potential unrelated donors (n=7) for non-Northwestern European patients, but almost doubled the number for Northwestern European patients from 42 to 71. Before and after 2007, an unrelated donor or cord blood was identified for 91% and 95%, respectively, of Northwestern European patients and for 65% and 82% of non-Northwestern European patients (P<0.0001). Non-Northwestern European patients more often needed a cord blood transplant. The degree of HLA matching was significantly lower for non-Northwestern European patients (P<0.0006). The time needed to identify a donor decreased for both populations. The percentage of Northwestern European patients reaching transplantation increased from 77% to 83% and for non-Northwestern European patients from 57% to 72% (P=0.0003). The increase of the global inventory resulted in more transplants for patients lacking a family donor, although the quality and quantity of (potential) haematopoietic cell grafts for patients of a non-Northwestern European descent remained inferior, indicating the need for adaptation of recruitment., (Copyright© Ferrata Storti Foundation.)

Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD15 have been demonstrated to modulate the outcome of allogeneic hematopoietic stem cell transplantation (SCT). The effect of NOD2/CARD15 polymorphism is reported to be associated with type of donor (sibling or matched unrelated donor) as well as type of conditioning regimen. We reviewed NOD2/CARD15 SNPs in all donor/recipient pairs of 192 consecutive patients who received nonmyeloablative allogeneic SCT at our institution between 2002 and 2006. All patients were treated with fludarabine 30 mg/m(2)/day for 3 days followed by 200 cGy total-body irradiation (TBI) (n = 154) or TBI alone (n = 38) and received grafts from HLA-matched related (n = 132) or unrelated (n = 61) donors. NOD2/CARD15 polymorphisms were observed in 36 of 192 (19%) patients and in 35 of 192 (18%) donors. The incidences of acute and chronic graft-versus-host disease (aGVHD, cGVHD) were 39% and 49%, respectively, in patients with NOD2/CARD15 variants versus 51% and 61% in patients with wild type. The relapse rate at 3 years was 38% in patients with variants and 36% in patients with wild type. The incidence of transplant-related mortality was 22% for patients with variants and 21% for patients with wild type. Overall survival (OS) at 3 years was 56% in patients with variants and 64% in patients with wild-type NOD2/CARD15. There was no significant impact of NOD2/CARD15 mutations on clinical outcome (all P > .05, Kaplan-Meier and Fine and Gray's tests). These data indicate that mutations in the NOD2/CARD15 gene are not a risk factor for clinical outcome in nonmyeloablative allogeneic SCT. Therefore, screening for NOD2/CARD15 polymorphisms in patients or donors does not have additional value in patients undergoing nonmyeloablative SCT., (Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Purpose: MHC class I chain related gene A (MICA), a polymorphic and stress-inducible cell surface molecule, is located centromeric to human leukocyte antigen locus B (HLA-B) in the human leukocyte antigen (HLA) region on chromosome 6. MICA is thought to be involved in the innate immune response. An alanine repeat polymorphism is present in the MICA transmembrane region, for which several disease associations have been reported. Previous research indicated an association with the HLA-B7-DR2 haplotype. In this study we investigated the association of the polymorphic MICA alanine repeat and the ocular disease presumed ocular histoplasmosis syndrome (POHS)., Methods: Twenty-four patients and 106 controls were evaluated for the alanine repeat. A PCR reaction was performed to amplify the polymorphic MICA alanine repeat. Allele lengths of the MICA alanine repeat in patients and controls were determined with GeneScan analysis., Results: No significant associations were observed. Phenotype frequencies of the polymorphic MICA alanine repeat were not significantly different between POHS patients and controls. Neither in the complete patient group compared with the control group nor in one of the subdivided patient groups compared with the control group., Conclusions: We conclude that the MICA alanine repeat is not a disease-associated factor in POHS. Further analysis of other genes in the B-DR region might elucidate the association of POHS with B7-DR2.

Purpose: To investigate whether presumed ocular histoplasmosis syndrome (POHS) in The Netherlands is associated with HLA-DR2 and HLA-B7, as previously shown in the United States., Methods: Twenty-four Dutch patients with POHS were included in this study. DNA isolated from peripheral blood leukocytes was typed for HLA by a sequence-based method. Associations were statistically determined. The frequencies of HLA alleles in bone marrow of donors listed on the European donor registry was used to represent the distribution in the normal population. Patients were included in the study only when no cells were present in the vitreous at any time and when fundus photographs fit the diagnosis made according to the following criteria: presence of peripapillary atrophy, presence of punched out chorioretinal lesions (histospots), and presence of a submacular scar. After the fundus photographs were judged, the patients were divided into two groups. Group1 contained patients who met all three diagnostic criteria (complete POHS), and group 2 contained patients who met one or two of the criteria (incomplete POHS)., Results: Group 1 consisted of 14 patients and group 2 of 10 patients. An association between POHS and HLA-DR2 and -B7 was present, compared with the normal Dutch control subjects. Although significant, the association between the frequency of HLA-DR2 and -B7 of all patients with POHS was less striking than the findings in patients with POHS in the United States. The association with DR2 in patients with incomplete POHS (group 2) was significantly different from that in the group with complete POHS (group 1). According to the defined criteria the association of POHS with HLA-B7 and -DR2 was confined to the incomplete POHS group and was not found in the complete POHS group. Furthermore, analysis of DR at the amino acid level, rather than at the allele level (DR2) showed that amino acid 67 of the DRB1 alleles had the most significant HLA association with POHS, independent of the two groups., Conclusions: POHS in Dutch patients was associated with HLA-B7 and -DR2, but more striking was the presence of isoleucine at position 67 of the HLA-DR molecule.