[Show abstract][Hide abstract] ABSTRACT: Improper activation of the immune system contributes to a variety of clinical conditions, including autoimmune and allergic diseases as well as solid organ and bone marrow transplantation. One approach to counteract this activation is through adoptive therapy with regulatory T cells (Tregs). Efforts to manufacture these cells have led to good maunfacturing practice–compliant protocols, and Treg products are entering early clinical trials. Here, we report the stance of the European Union Cooperation in Science and Technology Action BM1305, “Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies—A FACTT,” which identifies hurdles hindering Treg clinical applications in Europe and provides possible solutions.
Science translational medicine 09/2015; 7(304). DOI:10.1126/scitranslmed.aaa7721 · 15.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential of natural killer (NK) cells to target numerous malignancies in vitro has been well documented; however, only limited success has been seen in the clinic. Although NK cells prove non-toxic and safe regardless of the cell numbers injected, there is often little persistence and expansion observed in a patient, which is vital for mounting an effective cellular response. NK cells can be isolated directly from peripheral blood, umbilical cord blood, or bone marrow, expanded in vitro using cytokines or differentiated in vitro from hematopoietic stem cells. Drugs that support NK cell function such as lenalidomide and bortezomib have also been studied in the clinic, however, the optimum combination, which can vary among different malignancies, is yet to be identified. NK cell proliferation, persistence, and function can further be improved by various activation techniques such as priming and cytokine addition though whether stimulation pre- or post-injection is more favorable is another obstacle to be tackled. Here, we review the various methods of obtaining and activating NK cells for use in the clinic while considering the ideal product and drug complement for the most successful cellular therapy.
Frontiers in Immunology 06/2015; 6:264. DOI:10.3389/fimmu.2015.00264
[Show abstract][Hide abstract] ABSTRACT: Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.
PLoS ONE 05/2015; 10(5):e0127153. DOI:10.1371/journal.pone.0127153 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NK cells play a key role in innate elimination of virally infected or neoplastic cells but they can be circumvented by immunoevasive mechanisms enabling viral spread or tumour progression. Engagement of the NKG2D activating receptor with soluble forms of its ligand is one such mechanism of inducing NK cell hyporesponsiveness. Interestingly, this immunoevasive strategy among others is described at the maternal-fetal interface where tolerance of the semi-allogeneic fetus is required to allow successful human pregnancy. Understanding of maternal-fetal tolerance is increasing but mechanisms preventing alloreactivity of fetal immune cells against the maternal host are less well understood. The study of umbilical cord blood has enabled insight of the fetal immune system, which appears immature and inert. We have found that soluble NKG2D ligands (sNKG2DLs) are present in cord blood plasma (CBP) and associate with adult NK cell hyporesponsiveness demonstrated by reduced CD107a expression and secretion of IFN-γ upon stimulation. The capacity of NK cells to kill K562 cells or proliferate was also reduced by incubation with CBP; however, physical removal of sNKG2DL from CBP restored K562 lytic function and NKG2D expression. Therefore, our results strongly suggest sNKG2DLs are expressed in CBP as a mechanism of fetal-maternal tolerance in human pregnancy. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
European Journal of Immunology 05/2015; 45(8). DOI:10.1002/eji.201444990 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background and Objectives
Advantages of using cord blood (CB) over other sources of haematopoietic progenitor cells, such as bone marrow, include the ability to cryopreserve and bank the samples until requested for a transplant. Cryopreservation requires the addition of a cryoprotectant to prevent the formation of intracellular ice during freezing. Dimethyl sulphoxide (DMSO) is commonly used at a concentration of 10% (v/v); however, there is evidence to suggest this chemical is toxic to cells as well as to patients after infusion.Materials and Methods
The toxic effects of DMSO were assessed through cell viability and in vitro functional assays in fresh and post-thaw CB samples before determining the maximum exposure time and optimal concentration for cryopreservation.ResultsA dose-dependent toxicity of DMSO was observed in fresh samples with 40% removing all viable and functional haematopoietic progenitor cells (HPC). In fresh and post-thaw analysis, minimal toxic effect was observed when cryopreservation was delayed for up to 1 h after 10% DMSO addition. After thawing, DMSO washout was superior to dilution or unmanipulated when maintained for long periods (advantage observed 1 h after thawing). Finally, the optimum concentration for cryopreserving CB was found to be 7·5 to 10% with detrimental effects observed outside of this range.Conclusion
These results support the use of 7·5–10% as the optimal DMSO concentration and the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw.
[Show abstract][Hide abstract] ABSTRACT: Allo-SCT using unrelated donors is a curative treatment for patients with hematological disorders. The best donor is one matched for 10/10 HLA alleles, however studies have shown an additional survival benefit when considering other genetic factors. It has been shown that a six-nucleotide insertion/deletion polymorphism in the CASP8 gene promoter results in reduced susceptibility of T lymphocytes to undergo apoptosis. In 186 SCT recipients, we found a significantly better OS in those who received a transplant from a WT/WT donor compared with donors with a deletion (3 years: 52 vs 34%; P=0.03; multivariate analysis; RR 0.61; 95% CI 0.38-0.98, P=0.04). This was more marked when both the patient and the donor had a deletion (3 years OS: 62% compared with 36%, P=0.01). As the majority of these patients received Alemtuzumab during conditioning, we went on to analyze the in vitro effect of the polymorphism on Alemtuzumab-induced apoptosis. We showed statistically significantly higher percentages of apoptotic naïve CD4 (P<0.0005) and CD8 (P<0.0005) T cells in WT/WT donors in comparison with donors with a deletion. These data imply an unrecognized role for the CASP8 promoter polymorphism on survival following unrelated SCT particularly in the context of T-cell depletion with Alemtuzumab.Bone Marrow Transplantation advance online publication, 27 October 2014; doi:10.1038/bmt.2014.238.
Bone Marrow Transplantation 10/2014; 50(2). DOI:10.1038/bmt.2014.238 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HLA-B∗57:01 is a well-known and cost-effective pharmacogenetic marker for abacavir hypersensitivity. As with other HLA alleles, there is widespread variation in its frequency across populations. The Costa Rica Central Valley Population (CCVP) is the major population in this country. The frequency of HLA-B∗57:01 in this population has not been described yet. Thus, our aim was to determine the frequency of this allele in the CCVP. 200 unrelated healthy volunteer donors born in the CCVP were typed. HLA-B∗57-positive samples identified by HLA intermediate resolution typing methods were further typed by SBT to high resolution. An HLA-B∗57:01 carrier frequency of 5.00% was determined in this sample. This frequency is relatively high in comparison to reports from other populations in Latin America. These results suggest that there is a considerable frequency of HLA-B∗57:01 in the CCVP and that pharmacogenetic testing for HIV+ patients who are going to receive abacavir-based treatment should be considered in this country.
Human Immunology 10/2014; 75(11). DOI:10.1016/j.humimm.2014.09.011 · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are ligands for the natural killer group 2, member D (NKG2D) activating receptor expressed on natural killer (NK) cells, natural killer T (NKT) cells, CD8+ T cells and γδ T cells. Natural killer group 2, member D (NKG2D) ligand expression is stress-related and upregulated by infected or oncogenic cells leading to cytolysis. MICA and MICB genes display considerable polymorphism among individuals and studies have investigated allelic association with disease and relevance of MICA in transplantation, with variable success. It is now known that promoters of MICA and MICB are polymorphic with some polymorphisms associating with reduced expression. We sequenced International Histocompatibility Workshop (IHW) cell line DNA to determine promoter types and alleles encoded by exons 2-6. We found 8 of 12 known MICA promoter polymorphisms and although promoter P7 dominated, other promoters associated with the same allele. For example, MICA*002:01 had promoters P3, P4 or P7 and the common MICA*008:01/04 type had P1, P6 or P7. Similarly, we sequenced 8 of 12 known MICB promoter haplotypes. Some coding region defined MICB alleles had a single promoter, for example, MICB*002:01 and promoter P9, whereas the promiscuous MICB*005 allele had promoters P1, P2, P5, P6, P10 or P12. The results indicate potential for variation in expression of MICA and MICB ligands between individuals with the same allelic types. If differential expression by polymorphic MICA and MICB promoters is confirmed by functional studies, involvement of these genes in disease susceptibility or adverse transplantation outcomes may require knowledge of both promoter and allelic types to make meaningful conclusions.
[Show abstract][Hide abstract] ABSTRACT: Background
It is of clinical relevance to recognize donors who are unlikely to meet the requested stem cell dose for transplantation, as this group may benefit from an alternative mobilization regimen. This study was performed to evaluate the frequency of unrelated donor peripheral blood stem cell (PBSC) collections that meet the target yield and the impact of donor factors on this.Study Design and Methods
All sequential PBSC collections facilitated by the national registry (n = 323) from January through December 2011 were analyzed. Donor factors analyzed included age, sex, weight, and presence of a central line.ResultsIn univariate analyses, we found that reaching the target yield was significantly associated with a higher donor weight (85.6 kg vs. 75.3 kg, p < 0.001), male donor sex (55% vs. 19%, p < 0.001), a positive difference in weight between donor and recipient (4.3 kg vs. −8 kg, p < 0.001), and a higher volume of blood processed (13.8 L vs. 11.9 L, p < 0.001). After stepwise binary logistic regression, sex (p < 0.001) and difference between donor and recipient weight (p < 0.005) remained significantly associated with target yield being met after 1 day of collection.Conclusions
This study shows than women and donors who are lighter than their recipient have a decreased likelihood of meeting the transplant physician's requested dose. New strategies to improve mobilization in such donors are needed. These findings may also impact future donor recruitment strategies.
[Show abstract][Hide abstract] ABSTRACT: The selection of a related or an unrelated hematopoietic stem cell donor for a patient requires accurate matching of human leukocyte antigen (HLA) genes in order to maximize the beneficial effects of the transplant. There are various different factors a laboratory must consider in order to achieve an HLA type including the number of samples being processed, level of resolution to be achieved, cost of providing the various tests, and turnaround time required. Each method has its advantages and disadvantages, and in most laboratories, a combination of methods may be used.