Sonia Néron

CD40 Stimulation of Human Peripheral B Lymphocytes: Distinct Response from Naive and Memory Cells

During secondary immune response, memory B lymphocytes proliferate and differentiate into Ig-secreting cells. In mice, the binding of CD40 by CD154 clearly enhances the activation and differentiation of memory B lymphocytes. In humans, the role of CD40-CD154 in the stimulation of memory B lymphocytes is not as obvious since in vitro studies reported positive and negative effects on their proliferation and differentiation in Ig-secreting cells. In this study, we examine the response of peripheral memory and naive cells in relation to the duration of CD40-CD154 interaction. We measured the proliferation and differentiation of both subsets stimulated with CD154 and IL-4 for short- (4–5 days) and long-term (>7 days) periods. Following short-term stimulation, memory B lymphocytes did not expand but represented the only subset differentiating into IgG- and IgM-secreting cells. A longer stimulation of this population led to cell death, while promoting naive B lymphocyte proliferation, expansion, and differentiation into IgM- or IgG-secreting cells. This prolonged CD40 stimulation also triggered naive B lymphocytes to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent. This study suggests that naive and memory B lymphocytes have distinct requirements to engage an immune response, reflecting their different roles in humoral immunity.

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.

Tuning of CD40–CD154 Interactions in Human B-Lymphocyte Activation: A Broad Array of In Vitro Models for a Complex In Vivo Situation

Naive and memory B-lymphocyte populations can be activated through the binding of CD154 to CD40, a receptor that is constitutively expressed on the surface of these cells. Models based on the in vitro stimulation of human B lymphocytes through CD40 have greatly contributed to our understanding of the human immune response in healthy individuals and patients suffering from immune disorders. The nature of the engineered CD40 ligands is as diverse as the in vitro models used in studies of CD40-activated B lymphocytes. Monoclonal anti-CD40 antibodies, recombinant CD154 proteins, soluble CD154+ membranes as well as CD154+ cell lines have turned out to be very useful tools, and are still in use today. As for any receptor–ligand interaction, parameters such as duration and strength of contact, timing, affinity, and receptor density are major determinants of CD40 binding by CD154 or anti-CD40. Furthermore, variation in the intensity of CD40 stimulation has been shown to influence proliferation, differentiation and immunoglobulin secretion of human hybridomas, B-cell lines, tonsil and blood B lymphocytes. The objective of this review is to present an overview of the great diversity of CD40 agonists used in in vitro models of B-lymphocyte activation, with a particular emphasis on variations in the resulting strength of CD40 signaling generated by these models. A better understanding of these models could open up new avenues for the rational use of human B lymphocytes as antigen-presenting cells in cellular therapies.

Type 2 T‐Cell‐Independent Murine Immune Response to the Human AB0 Blood Group Antigens

The human AB0 blood group determinants are simple carbohydrate structures which are widely distributed in nature. Much work has been done on the structure of the A and B antigens but little is known on the regulation of anti‐A and anti‐B immune responses. To develop a model system, we have characterized the AB0 immunity of normal Balb/c mice and found a significant level of serum natural anti‐A but almost no anti‐B. This finding and the known IgM predominance among immune anti‐A produced in the Balb/c mouse indicate that the AB0 immunity of this mouse strain is comparable to the one of human blood group B individuals. Following immunization with human red blood cells, similar levels of anti‐A were produced in normal and athymic Balb/c mice showing that the anti‐A response is T cell independent. Furthermore, no anti‐A or anti‐B antibodies were produced in CBA/xid mice indicating a type 2 T‐cell‐independent immune response. These results may contribute to a better understanding of human AB0 responses and establish the mouse as a suitable model to study the immunobiology of AB0 antigens.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Whole‐blood leukoreduction filters are a source for cryopreserved cells for phenotypic and functional investigations on peripheral blood lymphocytes

BACKGROUND:  Leukoreduction of blood is now widely performed by blood banks, and the possibility of recovering 108 to 109 white blood cells (WBCs) from leukoreduction filters, which are usually discarded, represents a promising source for normal human cells. Previous studies with these filters to prepare WBCs have performed their experimentation with fresh cells only. Whether these filter‐derived cells could also be used to prepare frozen cell banks to facilitate work organization and open new avenues for their utilization as references in physiological studies and clinical investigations was investigated.

CD5+ B cell-dependent regulation of the murine T-cell independent immune response against the human blood group A antigen.

The CD5+B lymphocyte (B1a) population is known to be involved in most immune responses to microorganism TI antigens. Moreover, xid mice deficient for immune responses against TI-2 antigens are known to lack the B1a population, suggesting a role for B1a cells in TI-2 immune responses. We previously established that the oligosaccharide human blood group A antigen stimulated murine TI-2 immune responses. In this work, we show that the frequency of anti-A-secreting hybridomas was higher in mice with larger splenic B1a populations and that in vivo anti-CD5 treatment reduced anti-A immune response without affecting the response against TD RBC antigens. A similar effect was observed by in vitro anti-CD5 treatment of splenocytes. The in vivo anti-CD5 treatment also interfered with the immunization-dependent increase in splenocyte numbers. These results are in agreement with an important role for the B-cell CD5 receptor in the regulation of TI-2 immune responses possibly mediated by its interaction with the CD72 ligand.