Matching of HLA gene polymorphisms by high-resolution DNA sequence analysis is gold standard for ... more Matching of HLA gene polymorphisms by high-resolution DNA sequence analysis is 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 Human Leukocyte Antigen (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 1D2 nanopore reads tremendously. In the current study, we used 2 validation panels of samples covering 70 of the 71 known HLA class I allele groups to compare 3rd field sequences obtained by MinION with Sanger sequence-based typing (SSBT) 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 2nd validation panel. In summary, 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.
4322 Background Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD1... more 4322 Background Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD15 have been demonstrated to modulate the outcome of allogeneic haematopoietic stem cell transplantation. The effect of the NOD2/CARD15 polymorphism seems to be associated with donor source as well as type of conditioning regimen. Methods We reviewed NOD2/CARD15 mutations in all donor/recipient pairs of 192 consecutive patients who received non-myeloablative allogeneic stem cell transplantation(SCT) at our institution between 2002 and 2006. All patients were treated uniformly with fludarabine 30 mg/m2/day for 3 days followed by 200 cGy TBI (n=154) or TBI alone (n=38) and received grafts from HLA-matched related (n=132) or unrelated (n=60) donors. Results Mutated alleles were observed in 36 of 192 (19%) patients and in 35 of 192 (18%) donors. These SNPs, however, did not have a significant impact on clinical outcome data (P > 0.05, Kaplan Meier and Fine & Gray's test). Acute graft-v...
This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc... more This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Allocation to highly sensitized patients based on acceptable mismatches results in low rejection ... more Allocation to highly sensitized patients based on acceptable mismatches results in low rejection rates comparable to non-sensitized patients
by guest on 19 June 2022 is a risk marker for graft loss, whereas nDSAs in general are not associ... more by guest on 19 June 2022 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.
Multiplex Non-HLA Antibody Assay antigens coupled directly to MagPlex microspheres or indirectly ... more Multiplex Non-HLA Antibody Assay antigens coupled directly to MagPlex microspheres or indirectly via a HaloTag. The non-HLA antigens have been selected based on a literature search in patients with kidney disease or following transplantation. Due to the flexibility of the assay, this approach can be used to include alternative antigens and can also be used for screening of other organ transplant recipients, such as heart and lung.
Background Few studies have evaluated the effect of different immunosuppressive strategies on lon... more 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 u...
Natural killer (NK) cell-based immunotherapy is a promising novel approach to treat cancer. Howev... more Natural killer (NK) cell-based immunotherapy is a promising novel approach to treat cancer. However, NK cell function has been shown to be potentially diminished by factors common in the tumor microenvironment (TME). In this study, we assessed the synergistic potential of antibody-dependent cell-mediated cytotoxicity (ADCC) and killer immunoglobin-like receptor (KIR)-ligand mismatched NK cells to potentiate NK cell antitumor reactivity in multiple myeloma (MM). Hypoxia, lactate, prostaglandin E2 (PGE2) or combinations were selected to mimic the TME. To investigate this, NK cells from healthy donors were isolated and NK cell ADCC capacity in response to MM cells was assessed in flow cytometry-based cytotoxicity and degranulation (CD107a) assays in the presence of TME factors. Hypoxia, lactate and PGE2 reduced cytotoxicity of NK cells against myeloma target cells. The addition of daratumumab (anti-CD38 antibody) augmented NK-cell cytotoxicity against target cells expressing high CD38, but not against CD38 low or negative target cells also in the presence of TME. Co-staining for inhibitory KIRs and NKG2A demonstrated that daratumumab enhanced degranulation of all NK cell subsets. Nevertheless, KIR-ligand mismatched NK cells were slightly better effector cells than KIR-ligand matched NK cells. In summary, our study shows that combination therapy using strategies to maximize activating NK cell signaling by triggering ADCC in combination with an approach to minimize inhibitory signaling through a selection of KIR-ligand mismatched donors, can help to overcome the NK-suppressive TME. This can serve as a platform to improve the clinical efficacy of NK cells.
Routine typing of a sample from a donor joining the Australian Bone Marrow Donor Registry identif... more Routine typing of a sample from a donor joining the Australian Bone Marrow Donor Registry identified a potential novel HLA-C allele. The sample was initially typed using the One Lambda Labtype kit which identified a group 2 pair of common and well documented HLA-C alleles (one common allele and one not common allele): HLA-C ⁄ 06:02:01, 07:207. The laboratory routinely confirms 'non-common' alleles using sequence based typing. Exons 2 and 3 were sequenced in both directions using the Conexio SBT Resolver HLA-C kit and analysed using Assign SBT software. The sequencing data indicated an indel in exon 3, reflexive sequencing with HARPs identified the indel as a 36 bp insertion at position 592 of the HLA-C ⁄ 07 allele. Further analysis of the sequence indicated that the inserted 36 bp appeared to be a duplication of the previous 36 bp of the sequence in-frame, which would potentially add 12 amino acids to the mature protein. The sample was referred for NGS typing to our colleagues at the Department of Clinical Immunology, Fiona Stanley Hospital in Perth and was subsequently tested in-house using the Immucor Mia Fora NGS Flex kits. The NGS sequencing analysis results did not flag the presence of the insertion, calling the allele C ⁄ 07:18 with no unusual flags. Further analysis of the NGS data indicates that the software inserted bases to call the C ⁄ 07:18, which is unusual. This case study highlights a limitation of the NGS software packages to deal with large insertions. Although not conclusive to confirm this allele as having aberrant expression; the presence of the HLA-Cw7 antigen was not detected by serology using commercial typing trays.
Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocy... more Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendation...
NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK c... more NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLAclass I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR-ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy. NK cell • Multiple myeloma • HLA • Immunotherapy • KIR • NKG2A Abbreviations ADCC Antibody-dependent cellular cytotoxicity AML Acute myeloid leukemia BM Bone marrow Fab Fragment antigen binding GFP Green fluorescence protein GvHD Graft-versus-host disease HLA Human leukocyte antigen HSCT Hematopoietic stem cell transplantation KIR Killer immunoglobulin-like receptor MFI Mean fluorescence intensity MM Multiple myeloma NK Natural killer PCL Plasma cell leukemia Abstract Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-E low expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2 -/-γc -/-mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and
In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mut... more In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We enc...
Clinical and experimental immunology, Jan 30, 2014
Memory B-cells play a pivotal role in alloreactivity in kidney-transplantation. Follicular T-help... more Memory B-cells play a pivotal role in alloreactivity in kidney-transplantation. Follicular T-helper (TFH ) cells play an important role in the differentiation of B-cells into immunoglobulin-producing plasmablasts (through IL-21). It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney-transplant patients. Therefore we investigated the absolute numbers and function of peripheral TFH -cells (CD4(POS) CXCR5(POS) T-cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor specific anti-HLA antibodies (DSA), and the presence of TFH -cells in rejection biopsies. After transplantation, peripheral TFH -cell numbers remained stable, while their IL-21-producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral TFH -cells still had the capacity to induce B-cell differentiation and immunoglobulin production, which could be inhibited by an IL-21-receptor-a...
High resolution genotyping of human leukocyte antigen (HLA) class I and II alleles is important f... more High resolution genotyping of human leukocyte antigen (HLA) class I and II alleles is important for stem cell transplantation, however it is unclear whether polymorphisms outside of exon 2 and 3 are relevant. With the development of Sanger Sequence Based Typing (SSBT) methods, a high degree of polymorphism at the HLA loci has been identified, making allele identification challenging. Allele ambiguities frequently occur because of the inability to set phase and the lack of complete genomic sequences in reference databases. With the introduction of Next Generation Sequencing (NGS), it is now possible to sequence the HLA genes clonally, completely and simultaneously. Using in-house long range PCR-NGS protocols on the Ion PGM, the aim of this study was to confirm and obtain complete gene sequence for submission to the IMGT/HLA database, on samples identified as novel or rare for HLA Class I alleles by traditional short range (exons 2 and 3) SSBT. In addition, the full length sequences generated by NGS were confirmed using the novel full-length, group-specific sequence based typing (SBT) method, which results in hemizygous sequences, developed in Maastricht. For NGS, long range PCR which amplifies the full gene of HLA-A, -B and -C and exon 2 through to exon 3 of HLA Class II genes was performed. Long range amplicons for each individual are pooled for single library preparation using a modified version of the Ion PGM library preparation and 400 bp chemistry sequencing protocol. All data generated was analysed with software provided by Conexio Genomics and Life Technologies. Analysis of NGS-Ion PGM data identified and confirmed HLA Class I novel and rare alleles reported by short range SSBT. Furthermore, the group-specific sequence based typing results also confirmed data obtained by NGS-Ion PGM. In addition, a number of novel polymorphisms, mainly in intronic sequences, were identified by both sequence based typing methods. NGS-Ion PGM sequencing in addition to group-specific sequence based typing allowed the submission of several full length sequences to the IMGT/HLA database. This study shows the strength of NGS as a tool to complete the IMGT/HLA reference database and the continued relevance of SSBT as a reference methodology. This approach is currently been developed and applied to HLA Class II novel and rare alleles.
Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which pla... more Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.
Matching of HLA gene polymorphisms by high-resolution DNA sequence analysis is gold standard for ... more Matching of HLA gene polymorphisms by high-resolution DNA sequence analysis is 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 Human Leukocyte Antigen (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 1D2 nanopore reads tremendously. In the current study, we used 2 validation panels of samples covering 70 of the 71 known HLA class I allele groups to compare 3rd field sequences obtained by MinION with Sanger sequence-based typing (SSBT) 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 2nd validation panel. In summary, 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.
4322 Background Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD1... more 4322 Background Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD15 have been demonstrated to modulate the outcome of allogeneic haematopoietic stem cell transplantation. The effect of the NOD2/CARD15 polymorphism seems to be associated with donor source as well as type of conditioning regimen. Methods We reviewed NOD2/CARD15 mutations in all donor/recipient pairs of 192 consecutive patients who received non-myeloablative allogeneic stem cell transplantation(SCT) at our institution between 2002 and 2006. All patients were treated uniformly with fludarabine 30 mg/m2/day for 3 days followed by 200 cGy TBI (n=154) or TBI alone (n=38) and received grafts from HLA-matched related (n=132) or unrelated (n=60) donors. Results Mutated alleles were observed in 36 of 192 (19%) patients and in 35 of 192 (18%) donors. These SNPs, however, did not have a significant impact on clinical outcome data (P > 0.05, Kaplan Meier and Fine & Gray's test). Acute graft-v...
This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc... more This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Allocation to highly sensitized patients based on acceptable mismatches results in low rejection ... more Allocation to highly sensitized patients based on acceptable mismatches results in low rejection rates comparable to non-sensitized patients
by guest on 19 June 2022 is a risk marker for graft loss, whereas nDSAs in general are not associ... more by guest on 19 June 2022 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.
Multiplex Non-HLA Antibody Assay antigens coupled directly to MagPlex microspheres or indirectly ... more Multiplex Non-HLA Antibody Assay antigens coupled directly to MagPlex microspheres or indirectly via a HaloTag. The non-HLA antigens have been selected based on a literature search in patients with kidney disease or following transplantation. Due to the flexibility of the assay, this approach can be used to include alternative antigens and can also be used for screening of other organ transplant recipients, such as heart and lung.
Background Few studies have evaluated the effect of different immunosuppressive strategies on lon... more 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 u...
Natural killer (NK) cell-based immunotherapy is a promising novel approach to treat cancer. Howev... more Natural killer (NK) cell-based immunotherapy is a promising novel approach to treat cancer. However, NK cell function has been shown to be potentially diminished by factors common in the tumor microenvironment (TME). In this study, we assessed the synergistic potential of antibody-dependent cell-mediated cytotoxicity (ADCC) and killer immunoglobin-like receptor (KIR)-ligand mismatched NK cells to potentiate NK cell antitumor reactivity in multiple myeloma (MM). Hypoxia, lactate, prostaglandin E2 (PGE2) or combinations were selected to mimic the TME. To investigate this, NK cells from healthy donors were isolated and NK cell ADCC capacity in response to MM cells was assessed in flow cytometry-based cytotoxicity and degranulation (CD107a) assays in the presence of TME factors. Hypoxia, lactate and PGE2 reduced cytotoxicity of NK cells against myeloma target cells. The addition of daratumumab (anti-CD38 antibody) augmented NK-cell cytotoxicity against target cells expressing high CD38, but not against CD38 low or negative target cells also in the presence of TME. Co-staining for inhibitory KIRs and NKG2A demonstrated that daratumumab enhanced degranulation of all NK cell subsets. Nevertheless, KIR-ligand mismatched NK cells were slightly better effector cells than KIR-ligand matched NK cells. In summary, our study shows that combination therapy using strategies to maximize activating NK cell signaling by triggering ADCC in combination with an approach to minimize inhibitory signaling through a selection of KIR-ligand mismatched donors, can help to overcome the NK-suppressive TME. This can serve as a platform to improve the clinical efficacy of NK cells.
Routine typing of a sample from a donor joining the Australian Bone Marrow Donor Registry identif... more Routine typing of a sample from a donor joining the Australian Bone Marrow Donor Registry identified a potential novel HLA-C allele. The sample was initially typed using the One Lambda Labtype kit which identified a group 2 pair of common and well documented HLA-C alleles (one common allele and one not common allele): HLA-C ⁄ 06:02:01, 07:207. The laboratory routinely confirms 'non-common' alleles using sequence based typing. Exons 2 and 3 were sequenced in both directions using the Conexio SBT Resolver HLA-C kit and analysed using Assign SBT software. The sequencing data indicated an indel in exon 3, reflexive sequencing with HARPs identified the indel as a 36 bp insertion at position 592 of the HLA-C ⁄ 07 allele. Further analysis of the sequence indicated that the inserted 36 bp appeared to be a duplication of the previous 36 bp of the sequence in-frame, which would potentially add 12 amino acids to the mature protein. The sample was referred for NGS typing to our colleagues at the Department of Clinical Immunology, Fiona Stanley Hospital in Perth and was subsequently tested in-house using the Immucor Mia Fora NGS Flex kits. The NGS sequencing analysis results did not flag the presence of the insertion, calling the allele C ⁄ 07:18 with no unusual flags. Further analysis of the NGS data indicates that the software inserted bases to call the C ⁄ 07:18, which is unusual. This case study highlights a limitation of the NGS software packages to deal with large insertions. Although not conclusive to confirm this allele as having aberrant expression; the presence of the HLA-Cw7 antigen was not detected by serology using commercial typing trays.
Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocy... more Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendation...
NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK c... more NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLAclass I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR-ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy. NK cell • Multiple myeloma • HLA • Immunotherapy • KIR • NKG2A Abbreviations ADCC Antibody-dependent cellular cytotoxicity AML Acute myeloid leukemia BM Bone marrow Fab Fragment antigen binding GFP Green fluorescence protein GvHD Graft-versus-host disease HLA Human leukocyte antigen HSCT Hematopoietic stem cell transplantation KIR Killer immunoglobulin-like receptor MFI Mean fluorescence intensity MM Multiple myeloma NK Natural killer PCL Plasma cell leukemia Abstract Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-E low expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2 -/-γc -/-mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and
In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mut... more In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We enc...
Clinical and experimental immunology, Jan 30, 2014
Memory B-cells play a pivotal role in alloreactivity in kidney-transplantation. Follicular T-help... more Memory B-cells play a pivotal role in alloreactivity in kidney-transplantation. Follicular T-helper (TFH ) cells play an important role in the differentiation of B-cells into immunoglobulin-producing plasmablasts (through IL-21). It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney-transplant patients. Therefore we investigated the absolute numbers and function of peripheral TFH -cells (CD4(POS) CXCR5(POS) T-cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor specific anti-HLA antibodies (DSA), and the presence of TFH -cells in rejection biopsies. After transplantation, peripheral TFH -cell numbers remained stable, while their IL-21-producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral TFH -cells still had the capacity to induce B-cell differentiation and immunoglobulin production, which could be inhibited by an IL-21-receptor-a...
High resolution genotyping of human leukocyte antigen (HLA) class I and II alleles is important f... more High resolution genotyping of human leukocyte antigen (HLA) class I and II alleles is important for stem cell transplantation, however it is unclear whether polymorphisms outside of exon 2 and 3 are relevant. With the development of Sanger Sequence Based Typing (SSBT) methods, a high degree of polymorphism at the HLA loci has been identified, making allele identification challenging. Allele ambiguities frequently occur because of the inability to set phase and the lack of complete genomic sequences in reference databases. With the introduction of Next Generation Sequencing (NGS), it is now possible to sequence the HLA genes clonally, completely and simultaneously. Using in-house long range PCR-NGS protocols on the Ion PGM, the aim of this study was to confirm and obtain complete gene sequence for submission to the IMGT/HLA database, on samples identified as novel or rare for HLA Class I alleles by traditional short range (exons 2 and 3) SSBT. In addition, the full length sequences generated by NGS were confirmed using the novel full-length, group-specific sequence based typing (SBT) method, which results in hemizygous sequences, developed in Maastricht. For NGS, long range PCR which amplifies the full gene of HLA-A, -B and -C and exon 2 through to exon 3 of HLA Class II genes was performed. Long range amplicons for each individual are pooled for single library preparation using a modified version of the Ion PGM library preparation and 400 bp chemistry sequencing protocol. All data generated was analysed with software provided by Conexio Genomics and Life Technologies. Analysis of NGS-Ion PGM data identified and confirmed HLA Class I novel and rare alleles reported by short range SSBT. Furthermore, the group-specific sequence based typing results also confirmed data obtained by NGS-Ion PGM. In addition, a number of novel polymorphisms, mainly in intronic sequences, were identified by both sequence based typing methods. NGS-Ion PGM sequencing in addition to group-specific sequence based typing allowed the submission of several full length sequences to the IMGT/HLA database. This study shows the strength of NGS as a tool to complete the IMGT/HLA reference database and the continued relevance of SSBT as a reference methodology. This approach is currently been developed and applied to HLA Class II novel and rare alleles.
Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which pla... more Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.
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Papers by Marcel Tilanus