Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function.

The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins after human cytomegalovirus (HCMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hypermethylation. Genome-wide DNA methylation patterns were strikingly similar between HCMV-associated adaptive NK cells and cytotoxic effector T cells but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets.

Standardization of cellular therapy terminology, coding and labeling: a review.

The 1990s saw rapid growth in international activity in hematopoietic cell transplantation. As national donor registries were established and international collaboration increased, a need to transfer cellular therapy products across national borders emerged. A lack of international standards for identification, terminology and labeling resulted in significant challenges for import and export. Twenty years of effort by a large group of experts supported by professional societies and accreditation bodies has today achieved a high degree of standardization. This review highlights the main landmarks in this journey and serves as a reminder of the importance of taking the "long view" when working toward international standardization. It demonstrates the need for continual maintenance and enhancement of standards to meet the changing needs of the cell therapy industry and highlights recent developments in ISBT 128.

DEHT is a suitable plasticizer option for phthalate-free storage of irradiated red blood cells.

BACKGROUND AND OBJECTIVES: Due to increasing concerns about possible endocrine-disrupting properties, the use of the plasticizer di(2-ethylhexyl) phthalate (DEHP) will be banned in future blood storage. Di(2-ethylhexyl) terephthalate (DEHT) provides sufficient red blood cell (RBC) quality during conventional blood bank storage. It is important that a new plasticizer also maintains acceptable quality during exposure to high cell stress, such as irradiation, which is commonly used to prevent graft-versus-host disease. MATERIALS AND METHODS: A total of 59 RBC units were collected and processed in polyvinyl chloride (PVC)-DEHT or PVC-DEHP blood bags combined with either saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) additive solution. All units were X-ray irradiated on day 2 post-collection. Sampling for assessment of parameters of storage lesion was performed on day 2 pre-irradiation and day 14 and 28 post-irradiation. RESULTS: Though irradiation increased cell stress, DEHT/PAGGSM and current common European preference DEHP/SAGM were equally affected up to 14 days post-irradiation for all measured parameters. At day 28, haemolysis and microvesicle count were slightly increased in DEHT, whereas extracellular potassium ions, glucose, lactate, pH, mean corpuscular volume and microvesicle phosphatidylserine remained unaffected by plasticizer choice throughout storage. No individual unit exceeded 0.8% haemolysis, not even in DEHT/SAGM, the combination overall most affected by irradiation. Of the four combinations, membrane stability was least impacted in DEHP/PAGGSM. CONCLUSION: We demonstrate that DEHT is a suitable plasticizer for storage of RBCs after X-ray irradiation cell stress. This strengthens the option of DEHT as a viable non-phthalate substitute for DEHP.

Non-phthalate plasticizer DEHT preserves adequate blood component quality during storage in PVC blood bags.

BACKGROUND AND OBJECTIVES: Commercial blood bags are predominantly made of polyvinyl chloride (PVC) plasticized with di(2-ethylhexyl) phthalate (DEHP). DEHP is favourable for storage of red blood cells (RBC). Historically, removal of DEHP from blood bags has been linked to unacceptable haemolysis levels. Oncoming regulatory restrictions for DEHP due to toxicity concerns increase the urgency to replace DEHP without compromising RBC quality. Di(2-ethylhexyl) terephthalate (DEHT) is one suggested substitute. The aim of this study was to compare PVC-DEHT to PVC-DEHP blood bags using additive solutions saline-adenine-glucose-mannitol (SAGM) and phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM), to determine whether DEHT can maintain acceptable component quality. MATERIALS AND METHODS: RBC concentrates (N = 64), platelet concentrates (N = 16) and fresh frozen plasma (N = 32) were produced from whole blood collected into either DEHT or DEHP plasticized systems. Using a pool-and-split study design, pairs of identical RBC content were created within each plasticizer arm and assigned either SAGM or PAGGSM. Storage effects were assessed weekly for 49 days (RBC), 7 days (platelets) and before/after freezing (plasma). RESULTS: Though haemolysis was slightly higher in DEHT, all study arms remained below half of the European limit 0·8%. K+ was lower in DEHT than in DEHP independent of additive solution. The metabolic parameters were not influenced by choice of plasticizer. Platelet activation/metabolism and plasma content were similarly preserved. CONCLUSION: Our study demonstrates that the plasticizer DEHT provides adequate blood component quality. We propose DEHT as a strong future candidate for replacement of DEHP in blood bags.

A novel protocol for cryopreservation of paediatric red blood cell units allows increased availability of rare blood types.

BACKGROUND AND OBJECTIVES: There is a growing concern for shortage in future blood supply, caused by a predicted decrease in eligible blood donors and simultaneous increase in recipients. Cryopreservation of split red blood cell units could increase stock supply by reducing waste of rare blood. This would be particularly useful in paediatric care where very small volumes often are transfused. The aim of this study was to develop a cryopreservation protocol for split units using the closed-system automated cell processor ACP215, as such protocols are currently missing. MATERIALS AND METHODS: Using a pool-and-split design, red blood cell units (N = 8) were glycerolized and frozen, either as standard volume reference units, or further divided into three smaller split units each. After thawing/deglycerolization, the supernatant of the smaller splits was reduced by additional centrifugation, and new SAGM was added to 60% haematocrit. The units were analysed for storage lesion effects up to ten days post-thawing. RESULTS: Haemolysis and extracellular potassium ion levels were lower in the split units than in the whole units from day three onwards. The metabolic parameters pH, ATP, glucose and lactate were also lower, though likely caused by lower additive solution pH rather than storage. CONCLUSION: Split units of red blood cells can be successfully cryopreserved using the ACP215. In addition to favourably low haemolysis and potassium, they also have higher haematocrit than corresponding whole units and enable involvement of fewer donors at repeated transfusions. These characteristics are all desirable features in paediatric care.

Influence of KIR gene copy number on natural killer cell education.

Natural killer (NK) cells are functionally tuned by education via killer cell immunoglobulin receptors (KIRs) interacting with HLA class I molecules. We examined the effect of KIR gene copy number variation on the education of human NK cells. The frequency of NK cells expressing a given KIR correlated with the copy number of that gene. However, coexpression of multiple copies from a single locus, or duplicated loci, was infrequent, which is in line with independent transcriptional regulation of each allele or copy. Intriguingly, coexpression of 2 KIR alleles, resulting in higher surface expression, did not lead to enhanced functional responses in vitro or to selective advantages during in vivo responses to cytomegalovirus infection, suggesting that receptor density does not influence NK education at the single cell level. However, individuals with multiple KIR gene copies had higher frequencies of responding cells, consistent with heightened overall responsiveness.

CD8 T cells express randomly selected KIRs with distinct specificities compared with NK cells.

Epistatic interactions between killer cell immunoglobulin-like receptors (KIRs) and their cognate HLA class I ligands have important implications for reproductive success, antiviral immunity, susceptibility to autoimmune conditions and cancer, as well as for graft-versus-leukemia reactions in settings of allogeneic stem cell transplantation. Although CD8 T cells are known to acquire KIRs when maturing from naive to terminally differentiated cells, little information is available about the constitution of KIR repertoires on human CD8 T cells. Here, we have performed a high-resolution analysis of KIR expression on CD8 T cells. The results show that most CD8 T cells possess a restricted KIR expression pattern, often dominated by a single activating or inhibitory KIR. Furthermore, the expression of KIR, and its modulation of CD8 T-cell function, was independent of expression of self-HLA class I ligands. Finally, despite similarities in the stochastic regulation of KIRs by the bidirectional proximal promoter, the specificity of inhibitory KIRs on CD8 T cells was often distinct from that of natural killer cells in the same individual. The results provide new insight into the formation of KIR repertoires on human T cells.

Pathogen reduced red blood cells as an alternative to irradiated and washed components with potential for up to 42 days storage.

BACKGROUND: The urgency of maintaining a safe and adequate blood supply is increasing. One approach to ensure a sufficient supply is to limit the outdating frequency of blood components. Pathogen inactivation technology was developed primarily to increase safety by preventing transmission of infectious diseases. The Intercept Blood System for pathogen reduction of red blood cells (RBC) has additional benefits such as inactivation of leucocytes and removal of plasma and storage debris through centrifugation. Irradiation and automated washing are detrimental to the RBC membrane and often implicate shortened shelf-life. We aimed to assess whether pathogen inactivation can replace RBC irradiation and washing to avoid shelf-life reduction. MATERIALS AND METHODS: RBC concentrates (No.=48) were pooled-and-split into four study arms, which underwent pathogen inactivation treatment, irradiation, automated washing or no treatment (reference). RBC quality was evaluated during 42 days by assessment of storage lesion. Washing efficacy was defined by IgA and albumin reduction. RESULTS: Pathogen reduced RBCs had similar membrane preservation to reference RBCs (hemolysis, microvesicles and extracellular potassium ions), whereas the RBCs were negatively impacted by irradiation or automated washing. ATP increased substantially post-pathogen inactivation, while 2,3-DPG decreased. Pathogen inactivation considerably reduced albumin and IgA, though slightly less efficiently than automated washing. DISCUSSION: RBCs exhibit superior membrane preservation after pathogen inactivation treatment, compared to both irradiation and automated washing. This suggests that replacement is possible, even though the plasma reduction protocol could be further optimised.Replacement of irradiated and washed RBC concentrates with pathogen reduced RBC concentrates storable up to 42 days would be advantageous for both the blood supply and patient safety.

[Blood components, special components and whole blood - what and to whom?] / Blodkomponenter och helblod ­ så framställs och används de.

Sweden does not have a national blood authority and guidelines for blood transfusions are lacking, leading to varying routines of production and usage of blood in the different regions. The minimum quality requirements are defined in EU Directive 2002/98/EG and in the Swedish SOSFS 2009:28. The standard blood components are red blood cells, plasma and platelets, while special components such as irradiated, washed, frozen-thawed or antigen-matched products are prescribed on certain clinical indications. Thresholds for transfusion of red blood cells and platelets are discussed as well as indications for plasma transfusions. Further, there is evidence that early, balanced blood transfusions in massive bleeding reduce mortality, which has led to requests for blood products in prehospital settings.

Critical Role of CD2 Co-stimulation in Adaptive Natural Killer Cell Responses Revealed in NKG2C-Deficient Humans.

Infection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C (NKG2C(-/-)). Assessment of NK cell repertoires in 60 NKG2C(-/-) donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-γ promoter. We found that both NKG2C(-) and NKG2C(+) adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides "signal 2" in antibody-driven adaptive NK cell responses.

The effects of pneumatic tube transport on fresh and stored platelets in additive solution.

BACKGROUND: Limited scientific work has been conducted on potential in vitro effects of transport on pneumatic tube systems on blood components, in particular platelets. MATERIALS AND METHODS: To evaluate the possible effects of the Swisslog TranspoNet system on the cellular, metabolic, phenotypic and secreting properties of fresh and stored platelets, we set up a four-arm paired study comparing transported and non-transported platelets. Platelets were aliquoted, prepared with the OrbiSac system and suspended in 70% SSP+ (n=8). All in vitro parameters were monitored over a 7-day storage period. RESULTS: Throughout storage, no differences were observed in glucose consumption, lactate production, pH, pCO2, ATP, hypotonic shock response reactivity, CD62P, PAC-1, platelet endothelial cell adhesion molecule-1 or CD42b. The release of sCD40L increased (p<0.01) in all units but without any significant differences between groups. CONCLUSION: The storage stability of all platelets conveyed by the Swisslog TranspoNet system was not impaired throughout 7 days of storage. The Swisslog TranspoNet system does not, therefore, seem to be a risk for increased metabolic activity, activation or release reactions from the platelets. This lack of effect of the pneumatic tube transport system did not seem to be affected by the age of the platelets or repeated transport.

Do ABO blood group antigens hamper the therapeutic efficacy of mesenchymal stromal cells?

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

ISBT 128 implementation plan for cellular therapy products.

The publication of new standards for terminology and labeling marks an important step in ensuring consistency and traceability of cellular therapies at the global level. However, it is only with the widespread implementation of the standard that the benefits can be truly realized. This paper provides guidance on the practical aspects of adopting these new standards for organizations with differing current levels of computerization. It discusses project management, equipment, licensing, and validation topics.

Multicenter evaluation of a whole-blood filter that saves platelets.

BACKGROUND: Whole-blood (WB) leukoreduction filters in current use retain the majority of PLTs. A new whole-blood filter, which retains significantly fewer of the PLTs (or saves PLTs [WB-SP]), has been developed. The performance characteristics of the WB-SP filter have been evaluated in a multicenter study. STUDY DESIGN AND METHODS: A total of 617 units of WB was collected into quadruple bag sets with an integrated WB-SP filter, leukoreduced, and processed into leukoreduced RBCs (LR-RBC), plasma (LR-PL), and buffy coats (LR-BC) from which, pooled, leukoreduced, PLT concentrates (LR-PCs) were produced. Recovery, yield, and residual WBCs were assessed in prepared blood components. RESULTS: The median residual WBC number in the LR-RBCs was 0.05 x 10(6) (range, <0.05-3.8), exceeding 1 x 10(6) in 0.6 percent of the units. Median Hb content in LR-RBC was 50 g (range, 34-72), reflecting a final RBC recovery of 81 +/- 6 percent. The median WBC content of the LR-PC was 0.05 x 10(6) (range, <0.05-0.28), with none exceeding 1 x 10(6). The median PLT content of the LR-PC, per individual donation, was 6.4 x 10(10) (range, 4.1-10.7), representing a final recovery of 62 +/- 10 percent. The mean FVIII activity was 104 +/- 25 percent and 83 +/- 11 percent in plasma separated from fresh or overnight stored WB, respectively. CONCLUSION: Use of the WB-SP filter makes it possible to obtain three leukoreduced blood components with only one filtration step. The WB-SP filter showed good leukoreduction performance and recovery of all blood components including PLTs.