Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.

BACKGROUND AIMS: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. METHODS: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. RESULTS: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. CONCLUSIONS: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.

An objective flow cytometry method to rapidly determine cord blood potency in cryopreserved units.

BACKGROUND: Cord blood banks have to determine the regenerative potential of cord blood units (CBUs) on a representative sample of the cryopreserved product before release to the transplant center. Potency can be measured by using a colony-forming unit (CFU) method, which delays the release of CBU by 7 to 14 days. To accelerate CBU qualification, we have developed a rapid method to assess the response of CD34 cells to interleukin (IL)-3. Flow cytometry was used to measure IL-3-induced STAT5 phosphorylation within CD34-cells. This IL-3 test was compared to the CFU method, as well as the aldehyde dehydrogenase (ALDH) enzyme-based assay. STUDY DESIGN AND METHODS: Ten cryopreserved CBUs were analyzed for their contents in CD34 and CD45 viable cells, total CFUs, ADLHbright cells, and IL-3-responsive CD34+ cells. Extreme and mild warming event scenarios were simulated on CBUs and used as poor-quality samples. Segments, tubes, and bags from five CBUs were compared for their potency using IL-3 and CFU methods. RESULTS: The IL-3 test was accurate in identifying the samples handled following standard operating procedures and those subjected to extreme warming events. Based on these results, a threshold of 55% of IL-3-responsive CD34 cells was established to identify good-quality samples. The IL-3 test was also the most sensitive to detect samples subjected to milder warming events. CONCLUSIONS: Our new method for determining CBU functionality is rapid, unbiased, and robust. The IL-3 test described herein fulfills the requirements for validation, and we intend to implement this method in our cord blood bank facility.

Leukoreduction system chambers are a reliable cellular source for the manufacturing of T-cell therapeutics.

BACKGROUND: Following solid organ or hematopoietic cell transplantation, refractory opportunistic viral reactivations are a significant cause of morbidity and mortality but can effectively be controlled by virus-specific T-cell transfer. Among effective and safe strategies is the use of "third-party" (neither from the transplant donor nor recipient) virus-specific T cells that can be manufactured from healthy donors and used as "off-the-shelf" therapies. Leukoreduction system chambers (LRSCs), recovered after routine plateletpheresis, were evaluated as a potential source of peripheral blood mononuclear cells (PBMCs) for the manufacturing of clinical-scale virus-specific T cell. STUDY DESIGN AND METHODS: PBMCs from the same donors obtained either from LRSCs or peripheral blood were compared, focusing on T-cell function and phenotype as well as the potential to generate cytomegalovirus (CMV)-specific T-cell lines from both CMV seropositive and seronegative donors. RESULTS: PBMCs from both sources were comparable except for a transient downregulation of CD62L expression on freshly extracted PBMCs from LRSCs. Both nonspecific stimulation using anti-CD3/CD28 antibodies and CMV peptides revealed that LRSCs or blood T cells were equivalent in terms of expansion, differentiation, and function. Moreover, PBMCs from LRSCs can be used to generate autologous monocyte-derived dendritic cells to prime and expand CMV-specific T cells from seronegative donors. CONCLUSION: LRSCs are a reliable source of PBMCs for the generation of virus-specific T cells for immunotherapy. These findings have implications for the development of third-party therapeutic T-cell products from well-characterized blood product donors.

Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium.

The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC) from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50%) when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-ß, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium.

An alternative to dextran for the thawing of cord blood units.

BACKGROUND: Preparation of umbilical cord blood units (CBUs) for infusion requires a step of dilution or washing to reduce the toxicity of the dimethyl sulfoxide present in the freezing solution. However, the worldwide shortage of clinical-grade dextran 40, the most widely used cord blood dilution or washing solution, prompted us to search for an alternative solution. We elected to evaluate the performance of alternative solutions that could be used as potential replacements for the dextran 40-based solution. STUDY DESIGN AND METHODS: Frozen CBUs were rapidly thawed and immediately diluted 1:4 in each of 10 dilution solution variants of dextran 40, Plasma-lyte, or Hespan. We compared these alternatives by assessing viability, CD34, CD45, and colony-forming units recovery postthaw. RESULTS: A significantly lower CD34 and CD45 recovery was observed in all solutions without human serum albumin (HSA). All solutions with 5% HSA gave recovery, viability, and potency figures similar to the dextran 40/HSA solution. CONCLUSION: Based on our results and considering that Plasma-lyte A (PA)/HSA is already used for the thawing of CD34 from mobilized blood, we conclude that PA/HSA could be a safe and efficient solution for the replacement of dextran 40-based dilution solutions.

A global look into human T cell subsets before and after cryopreservation using multiparametric flow cytometry and two-dimensional visualization analysis.

The cryopreservation of human lymphocytes is an essential step for the achievement of several cellular therapies. Besides, T cells are considered as promising actors in cancer therapy for their cytotoxic and regulatory properties. Consequently, the development of tools to monitor the impact of freezing and thawing processes on their fine distribution may be an asset to achieve quality control in cellular therapy. In this study, the phenotypes of freshly isolated human mononuclear cells were compared to those observed following one cycle of cryopreservation and rest periods 0h, 1h and 24h after thawing but before staining. T cells were scrutinized for their distribution according to naive, memory effector, regulatory and helper subsets. Flow cytometry analyses were done using eight-color antibody panels as proposed by the Human Immunophenotyping Consortium. Data were further analyzed by using conventional directed gating and clustering software, namely SPADE and viSNE. Overall, SPADE and viSNE tools were very efficient to monitor the outcome of PBMC populations and T cell subsets. T cells were more sensitive to cryopreservation than other cells. Our results indicated that submitting the thawed cells to a 1h rest period improved the detection of some cell markers when compared to fresh samples. In contrast, cells submitted to a 24h rest period, or to none, were less representative of fresh sample distribution. The heterogeneity of PBMC, as well as the effects of freeze-thaw cycle on their distribution, can be easily monitored by using SPADE and viSNE.

In Vitro Culture of Human Hematopoietic Stem Cells in Serum Free Medium and Their Monitoring by Flow Cytometry.

Hematopoietic stem cells can be isolated from human blood cells trapped in leukoreduction systems. The leukoreduction systems filters or chambers are usually discarded from routine blood or platelet donations in blood banks around the world. These CD34+ cells are a good source of normal stem cells and can be used as models to characterize the blood stem cells before and after culture in vitro. This chapter contains detailed methodologies for the isolation of stem cells from peripheral blood, the culture of these cells in a medium exempt of animal proteins and for the flow cytometry analysis of the resulting cell population for the characterization of their differentiation.

Heterogeneity of in vitro-cultured CD34+ cells isolated from peripheral blood.

BACKGROUND AIMS: For transplantation, hematopoietic stem cells (HSC) are obtained from bone marrow, cord blood and mobilized adult peripheral blood. HSCs are present in the blood of healthy adults and can be recovered in leuko-reduction system chambers, with a potential yield of 1 to 4 × 10(6) CD34+ cells per unit. Some groups have investigated this valuable source of stem cells; however, investigations are still needed to support their use. METHODS: CD34+ cells were purified from leuko-reduction system chambers and cultured with a defined custom medium without animal protein and supplemented with interleukin-3, interleukin-6, Fms-like tyrosine kinase 3, stem cell factor and thrombopoietin. Cells were cultured under 8% and 21% oxygen levels. With the use of multiparametric flow cytometry analysis, the phenotypes of emerging populations were compared between oxygen levels and resting CD34+ cells. Both conventional gating and clustering analysis were used to visualize the cellular outcome. RESULTS: A maximum expansion of 20-fold was obtained without major differences in viability, number of cells or cellular heterogeneity between atmospheric and physiologic oxygen conditions. Worthy of note, phenotype analysis revealed that megakaryocyte and erythrocyte progenitors were favored, albeit more moderately when submitted to 8% O2. CONCLUSIONS: This study suggests that the bias of cultured blood CD34+ cells toward megakaryocyte and erythrocyte progenitor cells can be reduced by use of 8% pO2. It also shows how clustering software, such as SPADE, can help visualize the complexity of stem cell differentiation.

Rapid determination of IL-6 specific activity by flow cytometry.

Current methods to measure the specific activity of cytokines are based on the time-consuming determination of the growth curve of a sensitive cell line. Here, we present a faster alternative based on flow cytometry, by determining the dose-response curve of cellular response to a cytokine. By using World Health Organization (WHO) cytokine standards, rapid determination of cytokine specific activity is now possible, as it takes only a few hours to achieve, in comparison to days with the classical method thus allowing laboratories to rapidly and easily assess the potency of their cytokines.

N-acetyl cysteine regulates the phosphorylation of JAK proteins following CD40-activation of human memory B cells.

During their development, human B lymphocytes migrate into various environments, each presenting important variations in their redox balance depending on oxygen availability. The modulation of the cells surroundings redox balance leads to the regulation of reactive oxygen species produced by the cell. These molecules are involved in the state of oxidation of the cytosol and affect many pathways involved in cell development, differentiation and protein secretion. B lymphocytes cultured in presence of interleukin (IL)-2, IL-4, IL-10 and under CD154 stimulation, present increases in their intracellular levels of ROS. However, when N-acetyl cysteine (NAC), an antioxidant, is added, STAT3 phosphorylation is decreased. In this study, we show that in activated human memory B cells, NAC inhibited STAT3 phosphorylation on tyrosine 705 but not on Serine 727. Moreover, higher concentrations of NAC decreased STAT3 synthesis. Two other antioxidants, α-tocopherol and Trolox, did not affect STAT3 phosphorylation. Furthermore, two kinases involved in STAT3 activation, known as JAK2 and JAK3, appeared down-regulated in presence of NAC. In parallel, 3h after antioxidants incubation, we have observed a decrease in SOCS1 and SOCS3 protein levels, which seems time-related to antioxidant treatment. The decrease in the phosphorylation of JAK2 and JAK3, earlier in the process, could explain the downregulation of STAT3 and offer a hypothesis on the mechanism of action of NAC antioxidant properties which were confirmed by a decrease in the level of S-glutathionylation of proteins. The reduced expression of SOCS1 and SOCS3 appears directly linked to the inhibition of this STAT3-regulated pathway. In summary, NAC appears as a potential regulator of the STAT3 pathway.

Flow cytometry assessment of in vitro generated CD138+ human plasma cells.

The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs). As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98%) with variable stain index (SI). The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138(+) cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15-25%) while DL-101 mAb stained a higher proportion of CD138-positive cells (38-42%). DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138(high) and CD138(lo) cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

Comparison of the glycosylation of in vitro generated polyclonal human IgG and therapeutic immunoglobulins.

We have recently developed an in vitro culture model enabling the large-scale expansion of switched-memory B lymphocytes, producing a polyclonal human IgG repertoire. Given the importance of glycosylation for the functions of immunoglobulins, we analyzed the N-glycosylation profiles of the immunoglobulin G (IgG) in this model. Switched-memory B cells were cultured for 38 days and, using liquid chromatography-mass spectrometry, we analyzed IgGs' glycosylation profiles which were then compared to the glycosylation patterns of commercial intravenous immunoglobulin (IVIG). We observed a reproducible proliferation rate, high viability through the cultures as well as a good maintenance of the switched-memory B cells repertoire. The glycosylation pattern analyses revealed a variety of the typical biantennary N-glycan structures with diverse terminal monosaccharides. While many similarities were detected in comparison to the glycosylation profile of IVIG, in vitro-produced polyclonal IgGs were bearing higher levels of bisecting GlcNAc known to affect the effector functions of therapeutic antibodies. This data highlights the need for monitoring of the glycoform distribution in antibodies produced in vitro.

Feasibility study: phosphospecific flow cytometry enabling rapid functional analysis of bone marrow samples from patients with multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable cancer accounting for about 2% of cancer deaths. Its diagnosis is based on a combination of criteria, which are not always easily measurable. Flow cytometry now allows multiplex analysis of intracellular signaling at the single cell level. We investigated the feasibility of using intracellular protein phosphorylation analysis by flow cytometry on primary plasma cells from bone marrow and its usefulness in MM diagnosis. METHODS: Cells from frozen bone marrow of five MM patients and four normal donors were stimulated with LPS, IL-6, IL-21, IFNα and TNFα. Cells were stained by fluorescent cell barcoding to allow multiplex analysis. Staining with antibodies against phosphorylated NFkB-p65, Stat1, Stat3, and p38 were used to identify cellular responses following stimulation. RESULTS: Activation profiles of MM and normal plasma cells have been established. MM cells showed heterogeneous response profiles while normal cells responses were homogeneous between donors. We also noticed that many MM samples seemed to show elevated basal level of Stat3 phosphorylation. These results suggest that different response profiles in primary MM cells might correspond to different subtypes of the disease. Thus, we provide an example of how these results may be used as a criterion for MM subtypes classification. CONCLUSIONS: We demonstrate that flow cytometry can be used to study signaling pathways in primary MM cells. The heterogeneity observed in MM cells from different patients can prove valuable for MM characterization and represents an interesting avenue for future research in MM diagnosis.

Persistent Polyclonal B Cell Lymphocytosis B Cells Can Be Activated through CD40-CD154 Interaction.

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare disorder, diagnosed primarily in adult female smokers and characterized by an expansion of CD19(+)CD27(+)IgM(+) memory B cells, by the presence of binucleated lymphocytes, and by a moderate elevation of serum IgM. The clinical course is usually benign, but it is not known whether or not PPBL might be part of a process leading to the emergence of a malignant proliferative disorder. In this study we sought to investigate the functional response of B cells from patients with PPBL by use of an optimal memory B cell culture model based on the CD40-CD154 interaction. We found that the proliferation of PPBL B cells was almost as important as that of B cells from normal controls, resulting in high immunoglobulin secretion with in vitro isotypic switching. We conclude that the CD40-CD154 activation pathway is functional in the memory B cell population of PPBL patients, suggesting that the disorder may be due to either a dysfunction of other cells in the microenvironment or a possible defect in another B cell activation pathway.

Human CD38hiCD138⁺ plasma cells can be generated in vitro from CD40-activated switched-memory B lymphocytes.

B lymphocyte differentiation into long-lived plasma cells is the keystone event for the production of long-term protective antibodies. CD40-CD154 and CD27-CD70 interactions are involved in human B lymphocyte differentiation into CD38(hi)CD138(+) cells in vivo as well as in vitro. In this study, we have compared these interactions in their capacity to drive switched-memory B lymphocytes differentiation into CD38(hi)CD138(+) plasma cells. The targeted B lymphocytes were isolated from human peripheral blood, expanded for 19 days, and then submitted to CD70 or CD154 interactions for 14 days. The expanded B lymphocytes were constitutively expressing CD39, whereas CD31's expression was noticed only following the in vitro differentiation step (day 5) and was exclusively present on the CD38(hi) cell population. Furthermore, the generated CD38(hi)CD138(+) cells showed a higher proportion of CD31(+) cells than the CD38(hi)CD138(-) cells. Besides, analyses done with human blood and bone marrow plasma cells showed that in vivo and de novo generated CD38(hi)CD138(+) cells have a similar CD31 expression profile but are distinct according to their reduced CD39 expression level. Overall, we have evidences that in vitro generated plasma cells are heterogeneous and appear as CD39(+) precursors to the ones present in bone marrow niches.

Feasibility study: phospho-specific flow cytometry enabling rapid functional analysis of bone marrow samples from patients with multiple myeloma.

Background: Multiple myeloma (MM) is an incurable cancer accounting for about 2% of cancer deaths. Its diagnosis is based on a combination of criteria, which are not always easily measurable. Flow cytometry now allows multiplex analysis of intracellular signaling at the single cell level. We investigated the feasibility of using intracellular protein phosphorylation analysis by flow cytometry on primary plasma cells from bone marrow and its usefulness in MM diagnosis. Methods: Cells from frozen bone marrow of five MM patients and four normal donors were stimulated with LPS, IL-6, IL-21, IFN? and TNF?. Cells were stained by fluorescent cell barcoding to allow multiplex analysis. Staining with antibodies against phosphorylated NFkB-p65, Stat1, Stat3 and p38 were used to identify cellular responses following stimulation. Results: Activation profile of MM and normal plasma cells have been established. MM cells showed heterogeneous response profiles while normal cells responses were homogeneous between donors. We also noticed that many MM samples seemed to show elevated basal level of Stat3 phosphorylation. These results suggest that different response profiles in primary MM cells might correspond to different subtypes of the disease. Thus, we provide an example of how these results may be used as a criterion for MM subtypes classification. Conclusions: We demonstrate that flow cytometry can be used to study signaling pathways in primary MM cells. The heterogeneity observed in MM cells from different patients can prove valuable for MM characterization and represents an interesting avenue for future research in MM diagnosis. © 2013 Clinical Cytometry Society.

Large-scale in vitro expansion of polyclonal human switched-memory B lymphocytes.

Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients' accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10(6)-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG(1), IgG(2), IgG(3) and IgG(4) is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.

Modulation of CD40-activated B lymphocytes by N-acetylcysteine involves decreased phosphorylation of STAT3.

B lymphocyte activation, maturation and reshaping require the interaction of its receptor CD40 with its ligand CD154, which is expressed on activated T lymphocytes. Metabolism in activated B lymphocytes is also characterized with several REDOX changes including fluctuation of Reactive Oxygen Species (ROS). Herein, we first confirm that stimulation of human peripheral blood B lymphocyte with CD154 increases intracellular ROS level. Then, by treatments with two well-known antioxidants, N-acetylcysteine (NAC) and Trolox, we further investigate the influence of REDOX fluctuation in CD40-activated B lymphocyte homeostasis in long term culture (13 days). Treatments with NAC increase viability, decrease proliferation and Ig secretion and enhance homoaggregation of B lymphocytes while Trolox only induces a marginal increase of their Ig secretion. The NAC-induced homoaggregation phenotype is paralleled with increased expressions of CD54, CD11a, CD27 and CD38. Mechanistically, a 24h exposure of B lymphocytes with NAC is sufficient to show strong inhibition of STAT3 phosphorylation. Besides, the treatment of B lymphocytes with the STAT3 inhibitor VI increases viability and decreases proliferation and secretion as in NAC-treated cells thus showing a role for STAT3 in these NAC-induced phenotypes. This study done in a human-based model provides new findings on how REDOX fluctuations may modulate CD40-activated B lymphocytes during immune response and provide additional hints on NAC its immunomodulatory functions.

Effective in vitro expansion of CD40-activated human B lymphocytes in a defined bovine protein-free medium.

CD40-CD154 interaction is used to culture human B lymphocytes, which are now viewed as effectors to potentially promote T lymphocyte response against malignant cells in cell-based therapy. Currently, the media used, based on bovine serum, are raising concerns for patient safety in such therapy. In this study, we have investigated whether human B lymphocytes could be cultured in the absence of bovine serum. Blood CD19(+) B lymphocytes were activated using interaction through CD40 in medium containing defined animals or human proteins and lipids, and were monitored during short-term periods (≤15 days). Conventional stem-cell medium and a medium containing human albumin instead of bovine albumin were tested. We observed that the response of B lymphocytes appeared influenced by lot-to-lot variability in low density lipoproteins (LDL). Nevertheless, B lymphocyte proliferation and secretion in serum-free and bovine protein-free media were quite similar to that of cells cultured in medium containing FBS. Our results show that CD40-activated B lymphocytes can be cultured for up to 15 days in a serum-free medium containing human albumin, LDL, α-tocopherol and chemically-defined lipids.