scholarly journals An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization

Blood ◽  
2009 ◽  
Vol 114 (25) ◽  
pp. 5173-5181 ◽  
Author(s):  
Michel Jourdan ◽  
Anouk Caraux ◽  
John De Vos ◽  
Geneviève Fiol ◽  
Marion Larroque ◽  
...  
Keyword(s):  
B Cells ◽  
In Vitro Model ◽  
Plasma Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Memory B Cells ◽  
Vitro Model ◽  
Intermediate Cells ◽  
Activated B Cells

Abstract Human plasma cells (PCs) and their precursors play an essential role in humoral immune response but are rare and difficult to harvest. We report the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells in a 3-step and 10-day (D) culture, including a 6-fold cell amplification. We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells (activated B cells and plasmablasts) compared with memory B cells and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). We show this B cell–to-PC differentiation to involve IRF4 and AICDA expressions in D4 activated B cells, decrease of PAX5 and BCL6 expressions, and increase in PRDM1 and XBP1 expressions in D7 plasmablasts and D10 PCs. It involves down-regulation of genes controlled by Pax5 and induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). The detailed phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study PC dyscrasias, including multiple myeloma.

scholarly journals An in Vitro Model of Differentiation of Memory B Cells Into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1636-1636
Author(s):  
Michel Jourdan ◽  
Anouk Caraux ◽  
John De Vos ◽  
Geneviève Fiol ◽  
Marion Larroque ◽  
...  
Keyword(s):  
B Cells ◽  
In Vitro Model ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Memory B Cells ◽  
Vitro Model ◽  
Intermediate Cells

Abstract Abstract 1636 Poster Board I-662 Human plasma cells (PCs) and their precursors play an essential role in humoral immune response, but are rare and difficult to harvest. We report here i) the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells (MBCs) in a 3-step- and 10-day (D) culture, including a 6-fold cell amplification. ii) We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells - activated B cells (actBCs) and plasmablasts (PBs) - compared to MBCs and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). iii) We show this B cell to PC differentiation to involve IRF4 and AICDA expression in D4 actBCs, decrease of PAX5 and BCL6 expressions and increase in PRDM1 and XBP1 expressions in D7 PBs and D10 PCs. It involves downregulation of genes controlled by Pax5, induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). iv) The phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study plasma-cell dyscrasias, including Multiple Myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1051-1051
Author(s):  
Renee C Tschumper ◽  
Collin A Osborne ◽  
Pritha Chanana ◽  
Jaime I Davila ◽  
Denise K Walters ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
In Vitro Model ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Rna Seq ◽  
Repertoire Analysis ◽  
V Region ◽  
Vitro Model

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals The transcriptional landscape of the murine middle ear epithelium in vitro

2019 ◽  
Author(s):  
Apoorva Mulay ◽  
Md Miraj K Chowdhury ◽  
Cameron James ◽  
Lynne Bingle ◽  
Colin D Bingle
Keyword(s):  
Gene Expression ◽  
Middle Ear ◽  
In Vitro Model ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Inflammatory Cells ◽  
Differentially Expressed ◽  
Vitro Model ◽  
Transcriptional Landscape

AbstractOtitis media (OM) is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology, it is clear that epithelial abnormalities underpin the disease. The mechanisms underpinning epithelial remodelling in OM remain unclear. We recently described a novel in vitro model of mouse middle ear epithelial cells (mMEECs) that undergoes mucociliary differentiation into the varied epithelial cell populations seen in the middle ear cavity. We now describe genome wide gene expression profiles of mMEECs as they undergo differentiation. We compared the gene expression profiles of original (uncultured) middle ear cells, confluent cultures of undifferentiated cells (day 0 of ALI) and cells that had been differentiated for 7 days at an ALI. >5000 genes were differentially expressed among the three groups of cells. Approximately 4000 genes were differentially expressed between the original cells and day 0 of ALI culture. The original cell population was shown to contain a mix of cell types, including contaminating inflammatory cells that were lost on culture. Approximately 500 genes were upregulated during ALI induced differentiation. These included some secretory genes and some enzymes but most were associated with the process of ciliogenesis. Our in vitro model of differentiated murine middle ear epithelium exhibits a transcriptional profile consistent with the mucociliary epithelium seen within the middle ear. Knowledge of the transcriptional landscape of this epithelium will provide a basis for understanding the phenotypic changes seen in murine models of OM.

scholarly journals The transcriptional landscape of the cultured murine middle ear epithelium in vitro

Biology Open ◽  
2021 ◽  
Vol 10 (4) ◽  
Author(s):  
Apoorva Mulay ◽  
Md Miraj K. Chowdhury ◽  
Cameron T. James ◽  
Lynne Bingle ◽  
Colin D. Bingle
Keyword(s):  
Gene Expression ◽  
Middle Ear ◽  
In Vitro Model ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Inflammatory Cells ◽  
Differentially Expressed ◽  
Vitro Model ◽  
Transcriptional Landscape

ABSTRACT Otitis media (OM) is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology, it is clear that epithelial abnormalities underpin the disease. The mechanisms underpinning epithelial remodelling in OM remain unclear. We recently described a novel in vitro model of mouse middle ear epithelial cells (mMEECs) that undergoes mucociliary differentiation into the varied epithelial cell populations seen in the middle ear cavity. We now describe genome wide gene expression profiles of mMEECs as they undergo differentiation. We compared the gene expression profiles of original (uncultured) middle ear cells, confluent cultures of undifferentiated cells and cells that had been differentiated for 7 days at an air liquid interface (ALI). >5000 genes were differentially expressed among the three groups of cells. Approximately 4000 genes were differentially expressed between the original cells and day 0 of ALI culture. The original cell population was shown to contain a mix of cell types, including contaminating inflammatory cells that were lost on culture. Approximately 500 genes were upregulated during ALI induced differentiation. These included some secretory genes and some enzymes but most were associated with the process of ciliogenesis. The data suggest that the in vitro model of differentiated murine middle ear epithelium exhibits a transcriptional profile consistent with the mucociliary epithelium seen within the middle ear. Knowledge of the transcriptional landscape of this epithelium will provide a basis for understanding the phenotypic changes seen in murine models of OM.

scholarly journals A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Linda M. Lee ◽  
Hong Zhang ◽  
Karim Lee ◽  
Horace Liang ◽  
Alexander Merleev ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Low Frequencies ◽  
Almost All

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

MO251HIGHLY SENSITIVE FLOW CYTOMETRIC DETECTION OF RESIDUAL B-CELLS AFTER RITUXIMAB IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS PATIENTS*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y K O Teng ◽  
L Van Dam ◽  
Jelle Oskam ◽  
S W A Kamerling ◽  
E J Arends ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Peripheral Blood Mononuclear

Abstract Background and Aims B-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX. Method EuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA. Results By employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients. Conclusion We demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

Nurselike Cells in Chronic Lymphocytic Leukemia Have Gene Expression Profiles and Functional Characteristics That Are Distinct from Those of Monocytes or Dendritic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1183-1183
Author(s):  
Davorka Messmer ◽  
Tomoyuki Endo ◽  
Bradley T. Messmer ◽  
Danelle James ◽  
Nathan J. Zvaifler ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Mhc Ii ◽  
Blood Mononuclear Cells

Abstract When CD14+ blood mononuclear cells are cultured with chronic lymphocytic leukemia (CLL) B cells they can differentiate into “nurselike” cells (NLCs), which in turn can support the survival of CLL B cells in vitro and possibly in vivo. While factors that contribute to NLC-mediated support of CLL B cell survival have been identified, it is not clear how this cell type relates to other cell types that also can differentiate from CD14+ blood cells, such as monocyte-derived dendritic cells (DCs). Prior studies have identified some phenotypic differences between NLCs, DCs, and other CD14+ blood mononuclear cells. Thus we hypothesized that these cell types may have different gene expression patterns that may relate to distinctive functional properties. To resolve this we examined the genes expressed by monocytes, NLCs, and immature DCs using Affymetrix U133 microarray analyses. Gene expression profiles were generated from the CD14+ monocyte progenitors, NLC, and DC from three different individuals. The expression profiles of DCs and NLCs differed from the CD14+ progenitors by the expression of many thousands of genes and NLC were distinguished from DCs by the expression of several hundred genes. Some of the genes expressed at higher levels in DCs relative to NLCs encode accessory molecules involved in antigen presentation. Consistent with this, we found that immature DCs were 10 times more effective than NLCs in presenting antigen to allogeneic T cells. DCs express toll like receptors (TLR) on their cell surface that recognize pathogen components and upon exposure to TLR ligands DCs undergo a maturation process, whereby they upregulate surface molecules and gain increased T cell stimulatory capacity. The expression of TLR2, 4, and 9 was analyzed in DCs and NLCs by RT PCR. Both DCs and NLCs were found to express mRNA for TLR2 and 4, but only NLCs expressed TLR9. In concordance with this, NLCs but not DCs unregulated MHC-II after exposure to nonmethylated CpG oligodeoxinucleotides (ODN), a TLR9 agonist, whereas both cell types upregulated MHC-II after exposure to lipopolysaccharide. Given the propensity of CD14+ cells to differentiate down a NLC pathway when co-cultured with leukemic B cells in vitro, we speculate that differentiation of CD14+ cells into NLCs may be favored in patients with CLL over differentiation into DCs. Given the relative differences in APC function of these two cell types, this may account in part for the acquired immune deficiency often observed in patients with this disease. On the other hand a stimulus like CpG ODN, might increase the ability of NLCs to activate T cells and decrease their ability to support CLL B cells survival.

Tracking Factor VIII-Specific Memory B Cells in Hemophilia A Patients with Factor VIII Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1016-1016 ◽  
Author(s):  
Irene Lang ◽  
Jerzy Windyga ◽  
Anna Klukowska ◽  
Josenato Ilas ◽  
Hans Peter Schwarz ◽  
...  
Keyword(s):  
B Cells ◽  
Tetanus Toxin ◽  
Plasma Cells ◽  
Hemophilia A ◽  
Positive Control ◽  
Negative Control ◽  
Memory B Cells ◽  
Healthy Individuals ◽  
Specific Memory

Abstract The development of neutralizing anti-FVIII antibodies (FVIII inhibitors) in about 30% of patients with severe hemophilia A is the most serious complication in the treatment of hemophilia patients with FVIII products. Little information is available on the immunological mechanisms that regulate the development and maintenance of FVIII inhibitors. Memory B cells are a central component of humoral immunity. They drive the rapid anamnestic antibody response that occurs after re-exposure to antigen and seem to be important for replenishing the pool of long-lived plasma cells to maintain long-term antibody levels in the absence of antigen. Nothing is known about the dynamics of FVIII-specific memory B cells in patients with hemophilia A who develop FVIII inhibitors. Recently, Crotty et al. (J Immunol Methods, 2004) described an in vitro assay to quantify antigen-specific memory B cells in human blood. This assay utilizes a 6-day polyclonal stimulation of peripheral blood mononuclear cells (PBMC) followed by an antigen-specific ELISPOT for the detection of memory B cells that have differentiated into antibody-secreting plasma cells in vitro. We adapted this assay to human FVIII and used it to track FVIII-specific memory B cells in the blood of hemophilia A patients with and without FVIII inhibitors. Human serum albumin was used as a negative control and tetanus toxin as a positive control. The numbers of FVIII-specific, tetanus toxin-specific (positive control) and albumin-specific (negative control) memory B cells were calculated as percentage of total IgG memory B cells. So far, we have analyzed 14 patients with hemophilia A (age: 6–65 years). 8 were positive and 6 were negative for FVIII inhibitors. For comparison, we have analyzed 20 healthy individuals (age: 19–48 years). 2 out of 8 patients with inhibitors had detectable FVIII-specific memory B cells in their peripheral blood cells. However, none of the patients without inhibitors and none of the healthy individuals had any detectable FVIII-specific memory B cells in their circulation. The detection limit for FVIII-specific memory B cells in patients with inhibitors was about 0.2 % (percent of total IgG memory B cells). Current activities focus on further advancing the method with the aim to improve the detection limit for the detection of FVIII-specific memory B cells. All samples analyzed (including patients and healthy individuals) were negative for human serum albumin-specific memory B cells (negative control). Tetanus toxin-specific memory B cells (positive control) were found in both patients and healthy blood donors. The percentage of tetanus toxin-specific memory B cells in individuals who were vaccinated with tetanus toxoid was in the range of 0.25 – 0.58 % (percent of total IgG memory B cells). We conclude that the method described is suitable to track FVIII-specific memory B cells in the circulation. We are currently asking the question whether the presence of FVIII-specific memory B cells in the circulation correlates with the persistence of FVIII inhibitors. Furthermore, we will monitor patients with inhibitors during ITI therapy in order to find out whether the disappearance of FVIII-specific memory B cells in the circulation could be an early predictor of a successful ITI outcome.

Delayed Redistribution of CD27, CD40 and CD80 Positive B Cells and the Impaired In Vitro Immunoglobulin G Production in Patients with Non-Hodgkin Lymphoma Treated with Rituximab.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 938-938
Author(s):  
Mitsufumi Nishio ◽  
Katsuya Fujimoto ◽  
Satoshi Yamamoto ◽  
Toshiya Sakai ◽  
Kohki Kumano ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Healthy Controls ◽  
Immunoglobulin Production ◽  
Significant Difference ◽  
Healthy Control ◽  
Delayed Recovery

Abstract Rituximab (RT) has been proven to be very effective in depleting normal and malignant B lymphocytes in vivo and it is widely used for the treatment of B cell malignancies, particularly B cell non-Hodgkin’s lymphoma (NHL). RT alone does not appear to cause severe hypogammaglobulinemia according to initial clinical trials. However, recent studies revealed that patients who received RT as an adjuvant to stem cell transplantation (SCT) demonstrated an increased risk of developing severe hypogammaglobulinemia. We have found such hypogammaglobulinemia to be due to the delayed recovery of CD27 positive memory B cells and an impaired isotype expression. (Nishio et al. Eur J Haematol, 2006). This finding suggests that RT can influence not only the quantity, but also the quality of B-cell redistribution. Nevertheless, to our knowledge, precisely how the B-cell repertoire regenerates after anti-CD20-mediated transient B-cell depletion in patients with NHL remains to be elucidated. To clarify this, we performed a phenotypical analysis of B cells. A total of 22 patients with NHL who received RT combined with autologous SCT (n=17) or CHOP (n=5) were evaluated to identify their immunophenotype. The median period after the last administration of RT was 33.5 months (range from 12 to 56 months). We investigated the expression of various markers, including CD27, CD38, CD40, CD80, CD86 and CD95 on B cells by immunofluorescence staining with a flowcytometry analysis. A statistically significant difference was noted in three of the six surface antigens when the expressions of those antigens were compared with those in the healthy control populations (N=14). The most striking differences we found was the expression levels of CD27. The healthy control group had a much higher expression of CD27 in comparison to those of the patients treated with RT (28.1±14.1% vs 8.2±6.1%, p<0.001). In addition, significant differences in the expression of CD40 and CD80 were also noted. While the positive rates of CD80 and CD40 on B cells from healthy controls were 21.5±10.8% and 80.5±16.7%, those of patients treated with RT were 9.9±6.9% and 49.7±33.5%, respectively (p<0.01 and p<0.05). Since CD40-CD40L and CD80-CD28 pathways between B and T cells are necessary for the development of CD27 positive polyclonal B-cell activation and immunoglobulin production, we hypothesized that the B cells from patients treated with RT thus had a reduced ability to differentiate into plasma cells and immunoglobulin production in vitro. To test this hypothesis, we purified the B cells from ten patients with NHL treated with RT and then cultured them upon the engagement of immunoglobulin receptor and CD40 in the presence of IL-2 and IL-10. After eight days of stimulation, the supernatants of the culture were harvested and the concentrations of immunoglobulin were measured by ELISA. As a result, the IgG production was found to be significantly impaired in patients with NHL in comparison to those from the healthy controls. The observation of a delayed recovery of the memory B cells with an abnormal cell marker expression and function demonstrates that naive B cells may therefore be responsible for their failure to differentiate into plasma cells after RT therapy.

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