Annie Roy

Differential responses of human B-lymphocyte subpopulations to graded levels of CD40–CD154 interaction

Naïve and memory B‐lymphocyte populations are activated by CD154 interaction through cell‐surface CD40. This interaction plays an important role in the regulation of the humoral immune response, and increasing evidence indicates that fine variation in CD40 binding influences B lymphocytes, macrophages and dendritic cells in murine models. Here we have investigated whether and how variations in the intensity of the CD40–CD154 interaction could contribute to differential regulation of human B‐lymphocyte populations. Proliferation and differentiation of B lymphocytes were monitored in response to graded levels of CD40 stimulation in the presence of interleukin (IL)‐2, IL‐4 and IL‐10. Our results show that the level of CD154 binding to CD40 on B lymphocytes can directly influence the evolution of CD19+ CD27– and CD19+ CD27+ cell populations. Furthermore, proliferation, global expansion of CD19+ cells and emergence of CD38++ CD138+ cells, as well as immunoglobulin G (IgG) and IgM secretion, were affected by the level of exposure of B lymphocytes to CD154. These results suggest that the CD40–CD154 interaction is more like a rheostat than an on/off switch, and its variation of intensity may play a role in the regulation of B‐lymphocyte activation following the primary and/or secondary humoral immune response.

Characterization of mononuclear cells remaining in the leukoreduction system chambers of apheresis instruments after routine platelet collection: a new source of viable human blood cells

BACKGROUND: The yield of white blood cells (WBCs) extracted from whole‐blood leukoreduction filters can be affected by the storage conditions and delay before filtration. Platelets (PLTs) collected with apheresis instruments (Trima Accel, Gambro BCT) are leukoreduced during the procedure on a fluidized particle bed in a leukoreduction chamber (LRS chamber). In this report, the residual cell content of these LRS chambers was characterized to determine whether it would be a valuable source of viable human blood cells.

Increased Efficiency of γ-Irradiated versus Mitomycin C-Treated Feeder Cells for the Expansion of Normal Human Cells in Long-Term Cultures

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

Peripheral blood CD27+ IgG+ B cells rapidly proliferate and differentiate into immunoglobulin-secreting cells after exposure to low CD154 interaction

In vitro CD40 stimulation of human B cells isolated from lymphoid organs is dominated by memory B cells undergoing faster proliferation and higher differentiation than naive B cells. In contrast, we previously reported that blood memory B cells mainly differentiate into immunoglobulin‐secreting cells in response to CD40 stimulation. However, variations in CD40–CD154 interaction are now recognized to influence B‐cell fate. In this study, we have compared the in vitro response of blood CD27− and CD27− IgG− to CD27+ and CD27+ IgG+ B cells following low‐density exposure to CD154 in the presence of a mixture of interleukin‐2 (IL‐2), IL‐4 and IL‐10. The evolution of these cell populations was monitored during initiation and following long‐term stimulation. Over a 5‐day period, CD27+ B cells underwent differentiation into immunoglobulin‐secreting cells more readily than CD27− cells, and CD27+ IgG+ B cells gave rise to a near homogeneous population of CD19+ CD27++ CD38+ IgGlo cells capable of high immunoglobulin G (IgG) secretion. During the same period, CD27− IgG− B cells partially became CD19++ CD27− CD38− IgG++ cells but showed no IgG secretion. Long‐term stimulation revealed that CD27+ IgG+ B cells retained a high expansion capacity and could maintain their momentum towards differentiation over naive B cells. In addition, long‐term stimulation was driving CD27− IgG− and total CD19+ B cells to evolve into similar CD27+ and CD27− subsets, suggesting naive homeostatic proliferation. Overall, these results tend to reconcile memory B cells from blood and lymphoid organs regarding their preferential differentiation capacity compared to naive cells, and further suggest that circulating memory IgG+ cells may be intrinsically prone to rapid activation upon appropriate stimulation.

TELOMERE-INDEPENDENT REDUCTION OF HUMAN B LYMPHOCYTE: PROLIFERATION DURING LONG-TERM CULTURE

Telomeres and telomerase, the telomere lengthening enzyme, have been shown to play a central role in the long-term ability of cells to proliferate and maintain viability. In opposition to transformed cells, normal somatic cells express a low level of telomerase, which results in the gradual shortening of their telomeres after each division and in cell senescence once a critical telomere length is reached. We have tested the hypothesis that shortening of telomeres could limit the expansion of normal human B lymphocytes maintained in long-term culture using a CD40/CD154 system. Measurement of temolerase activity in cell lysates showed a rapid up-regulation of telomerase following the initiation of the culture that was dependent on the CD40 signaling. The high level of telomerase activity and the corresponding long telomere structures remained constant for the 35 day culture period in which a gradual reduction of the cell expansion rate is observed. We conclude that the gradual in vitro senescence of cultured B cells does not correlate with a corresponding loss of telomerase activity and of telomere length. Rather the phenomenon may be related to an intrinsic property of the proliferating B cells to differentiate into Ig-secreting cells.

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 1×106-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG1, IgG2, IgG3 and IgG4 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.

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.

Estimation of the number of CD154 molecules in membrane extracts used as a source of CD40 stimulation of human B lymphocytes

The CD40-CD154 interaction is better exemplified by a rheostat than by an on-off switch, and variations in its intensity can play a role in the regulation of B lymphocyte activation following primary and/or secondary humoral immune response. The CD40-CD154 interaction is often studied in co-culture models using CD154+ adherent cells, which can be problematic when performing protein or gene analyses. The use of membrane extracts prepared from CD154+-transfected cells can eliminate possible interferences caused by the presence of contaminating feeder cells. Given the dose-response effect of CD154 on target B cells, it is important to measure the amount of CD154 when using soluble membranes. We hereby report a simple method, based on cytometry analysis, to estimate the relative number of CD154 molecules in membrane extracts, allowing reproducibility in human B-cell activation level.

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.