Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells.

Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+ isolation, and a 3 × 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117-DRAQ5- RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.

Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile.

Non-small cell lung cancer (NSCLC) is the second most prevalent type of cancer. With the current treatment regimens, the mortality rate remains high. Therefore, better therapeutic approaches are necessary. NSCLCs generally possess many genetic mutations and are well infiltrated by T cells (TIL), making TIL therapy an attractive option. Here we show that T cells from treatment naive, stage I-IVa NSCLC tumors can effectively be isolated and expanded, with similar efficiency as from normal lung tissue. Importantly, 76% (13/17) of tested TIL products isolated from NSCLC lesions exhibited clear reactivity against primary tumor digests, with 0.5%-30% of T cells producing the inflammatory cytokine Interferon (IFN)-γ. Both CD4+ and CD8+ T cells displayed tumor reactivity. The cytokine production correlated well with CD137 and CD40L expression. Furthermore, almost half (7/17) of the TIL products contained polyfunctional T cells that produced Tumor Necrosis Factor (TNF)-α and/or IL-2 in addition to IFN-γ, a hallmark of effective immune responses. Tumor-reactivity in the TIL products correlated with high percentages of CD103+CD69+CD8+ T cell infiltrates in the tumor lesions, with PD-1hiCD4+ T cells, and with FoxP3+CD25+CD4+ regulatory T cell infiltrates, suggesting that the composition of T cell infiltrates may predict the level of tumor reactivity. In conclusion, the effective generation of tumor-reactive and polyfunctional TIL products implies that TIL therapy will be a successful treatment regimen for NSCLC patients.

A Homozygous Mutation on the HBA1 Gene Coding for Hb Charlieu (HBA1: c.320T>C) Together with ß-Thalassemia Trait Results in Severe Hemolytic Anemia.

A 4-year-old boy, a ß-thalassemia (ß-thal) carrier, with an unexplained severe chronic microcytic anemia was referred to us. Sequencing of the α-globin genes revealed a Hb Charlieu [α106(G13)Leu→Pro, HBA1: c.320T>C, p.Leu107Pro] mutation present on both HBA1 genes. Quantitative polymerase chain reaction (qPCR) confirmed αCharlieu mRNA in the proband and his parents, showing that the mutation does not affect mRNA stability. However, we were unable to detect the Hb Charlieu protein by capillary electrophoresis (CE), reverse phase electrophoresis, cation exchange electrophoresis or isoelectric focusing. Mass spectrometry (MS) allowed us to confirm the presence of the Hb Charlieu peptide in erythrocyte progenitors. These findings suggest that the mutation affects the stability of αCharlieu. As hemoglobin (Hb) heat stability tests showed no abnormalities in erythrocytes, we speculated that αCharlieu is already degraded during red blood cell (RBC) development. The clinical severity in the proband and the presence of new methylene blue-stained aggregates in his reticulocytes indicates that incorporation of αCharlieu destabilizes Hb. This, combined with an excess of unstable free α-globins as the result of ß-thal minor, results in severely impaired erythropoiesis and, as a consequence, severe and chronic microcytic anemia in the proband.

Potassium leakage primes stored erythrocytes for phosphatidylserine exposure and shedding of pro-coagulant vesicles.

During storage, erythrocytes undergo changes that alter their clearance and function after transfusion and there is increasing evidence that these changes contribute to the complications observed in transfused patients. Stored erythrocytes were incubated overnight at 37°C to mimic the temperature after transfusion. After incubation, several markers for erythrocyte damage were analysed. After overnight incubation, stored erythrocytes showed increased potassium leakage, haemolysis, PS exposure and vesicle formation, and all these effects increased with increasing storage time. Furthermore, we demonstrated that long-term stored erythrocytes develop decreased flippase activity and increased scrambling activity after overnight incubation, leading to PS exposure and the release of vesicles. Reduced intracellular potassium was identified as the cause of the decreased flippase activity. Lastly, we provide evidence that erythrocytes can return to a PS-negative state by shedding parts of their membrane as PS-containing vesicles and that these vesicles can serve as a platform for the coagulation cascade. These findings reveal that potassium leakage, a well-known phenomenon of prolonged erythrocyte storage, primes erythrocytes for PS exposure. PS exposure will lead to vesicle formation and might have an important impact on the post-transfusion function and side effects of stored erythrocytes.

CD47 functions as a molecular switch for erythrocyte phagocytosis.

CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRPα expressed by macrophages. Thus, the CD47-SIRPα interaction constitutes a negative signal for erythrocyte phagocytosis. However, we report here that CD47 does not only function as a "do not eat me" signal for uptake but can also act as an "eat me" signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47-SIRPα interactions. Furthermore, we provide evidence that experimental aging of erythrocytes induces a conformational change in CD47 that switches the molecule from an inhibitory signal into an activating one. Preincubation of experimentally aged erythrocytes with human serum before the binding assay was required for this activation. We also demonstrate that aged erythrocytes have the capacity to bind the CD47-binding partner thrombospondin-1 (TSP-1) and that treatment of aged erythrocytes with a TSP-1-derived peptide enabled their phagocytosis by human red pulp macrophages. Finally, CD47 on erythrocytes that had been stored for prolonged time was shown to undergo a conformational change and bind TSP-1. These findings reveal a more complex role for CD47-SIRPα interactions in erythrocyte phagocytosis, with CD47 acting as a molecular switch for controlling erythrocyte phagocytosis.

Collection and storage of red blood cells with anticoagulant and additive solution with a physiologic pH.

BACKGROUND: A donation of whole blood is most commonly collected in acidic citrate-phosphate-dextrose (CPD) variants with pH 5.2 to 6.2 as anticoagulants. Previously, we have shown that the initial pH after red blood cell (RBC) preparation can have an effect on RBCs during storage. First, we investigated the effect of the pH of the anticoagulant on RBCs. Second, we investigated the possibility of decreasing the pH of our new additive solution (AS) phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) from pH 8.2 to 7.4 in combination with an anticoagulant with a physiologic pH. STUDY DESIGN AND METHODS: Whole blood was collected in CPD (pH 5.6) or trisodiumcitrate (TNC; pH 7.4), and leukoreduced units were prepared using saline-adenine-glucose-mannitol as AS. Second, whole blood was collected in TNC (pH 7.4), and leukoreduced units were prepared using PAGGGM (pH 7.4) or PAGGGM (pH 8.2) as AS. During cold storage, several in vitro characteristics were analyzed. RESULTS: In agreement with our previous findings, the initial pH of whole blood has an effect during storage of RBCs. In the second part we show that there are no differences between PAGGGM (pH 7.4) and PAGGGM (pH 8.2) units when an anticoagulant with a physiologic pH was used. CONCLUSION: These results indicate that the pH of the anticoagulant used during whole blood collection has an effect during storage of RBCs. When an anticoagulant with a physiologic pH is used during whole blood collection, the pH of PAGGGM can be decreased to physiologic levels, while maintaining adenosine triphosphate and 2,3-diphosphoglycerate levels.

CD47 in Erythrocyte Ageing and Clearance - the Dutch Point of View.

Recently, an important role for CD47, a well-known 'don't eat me' signal, in the clearance of aged erythrocytes was revealed. Experimental data support the conversion of CD47 from a 'don't eat me' to an 'eat me' signal through a conformational change in CD47. Intriguingly, erythrocyte phagocytosis after this switch seems to be mediated by the same receptor that normally signals inhibition of phagocytosis, SIRPα. In this review, the possible molecular mechanisms leading to this conformational change in CD47 as well as the possible signal transduction events leading to phagocytosis after this switch are discussed. Lastly, the consequences of this newly identified mode of erythrocyte phagocytosis for the clearance of aged erythrocytes during normal turnover and after erythrocyte transfusion are addressed.

An improved red blood cell additive solution maintains 2,3-diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage.

BACKGROUND: Current additive solutions (ASs) for red blood cells (RBCs) do not maintain constant 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) levels during cold storage. We have previously shown that with a new AS called phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM), both 2,3-DPG and ATP could be maintained throughout storage for 35 days. STUDY DESIGN AND METHODS: In this study, the mechanism underlying the effect of PAGGGM on RBC storage was studied in more detail. By using double-erythrocytapheresis units (leukoreduced), a direct comparison could be made between the current AS saline-adenine-glucose-mannitol (SAGM) and the experimental solution PAGGGM. During cold storage, several in vitro characteristics were analyzed. RESULTS: In agreement with our previous findings with single RBCs, PAGGGM maintained 2,3-DPG and ATP levels for 35 days of cold storage. Furthermore, glucose consumption and lactate production were higher in PAGGGM units during the first 21 days of cold storage. Fructose-1,6-diphophate and dihydroxyacetone phosphate levels were also increased during the first 21 days of storage in PAGGGM units. CONCLUSION: These results indicate that it is likely that phosphofructokinase (PFK) activity is enhanced in PAGGGM units relative to SAGM units. After 21 days, PFK activity also decreases in PAGGGM units, but sufficient metabolic reserve in these units prevents depletion of 2,3-DPG and ATP.