scholarly journals CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and facilitates apoptosis through the Apo-1/Fas pathway.

1995 ◽  
Vol 182 (5) ◽  
pp. 1557-1565 ◽  
Author(s):  
E J Schattner ◽  
K B Elkon ◽  
D H Yoo ◽  
J Tumang ◽  
P H Krammer ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
B Cells ◽  
Programmed Cell Death ◽  
B Cell ◽  
B Lymphocytes ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Cd40 Ligation

The Apo-1/Fas antigen (CD95) mediates programmed cell death of lymphocytes when bound by Fas ligand or anti-Apo-1/Fas antibody. In contrast, the CD40 antigen provides a potent activation and survival signal to B lymphocytes when it is engaged by its T cell ligand (CD40L, gp39) or cross-linked by anti-CD40 antibody. In this study, we use human tonsillar B cells and the Ramos Burkitt's lymphoma B cell line, which serves as a model for human germinal center B lymphocytes, to study the effectors of Apo-1/Fas expression and apoptosis of human B cells. We found that Apo-1/Fas expression was upregulated on both malignant and normal human B lymphocytes after CD40 ligation induced by (a) cognate T helper-B cell interaction mediated by microbial superantigen (SAg); (b) contact-dependent interaction with CD40L+, but not CD40L- Jurkat mutant T cell clones; and (c) monoclonal anti-CD40, but not any of a panel of control antibodies. Enhanced B cell Fas/Apo-1 expression is functionally significant. Coculture of Ramos Burkitt's lymphoma line cells with irradiated SAg-reactive CD4+ T cells with SAg or CD40L+ Jurkat T cells results in B cell apoptosis, evidenced by reduced cell viability and DNA laddering. This process is augmented by the addition of anti-Apo-1/Fas monoclonal antibody, consistent with an acquired susceptibility to Apo-1/Fas-mediated apoptosis. These data support an immunoregulatory pathway in which seemingly contradictory signals involving the B cell proliferation/survival antigen CD40, as well as the Apo-1/Fas molecule, which mediates programmed cell death of lymphocytes, are linked in the process of human B cell activation.

scholarly journals CD4+ T-cell induction of Fas-mediated apoptosis in Burkitt's lymphoma B cells

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1375-1382 ◽  
Author(s):  
EJ Schattner ◽  
J Mascarenhas ◽  
J Bishop ◽  
DH Yoo ◽  
A Chadburn ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Fas Ligand ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Cd4 T Cell ◽  
Cd40 Ligation ◽  
Death Signals

Cytotoxic function of CD4+ Th1 cells is mediated by Fas (CD95, APO-1) and its ligand (Fas ligand). Recent studies using nontransformed B cells and the Ramos Burkitt's lymphoma (BL) B-cell line cells show that CD40 ligation at the B-cell surface by activated, CD40 ligand (CD40L)- bearing, CD4+ T cells upregulates Fas expression on B cells and primes B cells for Fas-mediated death signals. In this work, we examine whether this CD4+ T-cell-dependent molecular pathway for Fas upregulation and B-cell apoptosis reflects a peculiarity of the Ramos B- cell line or is applicable to other Burkitt's tumors as well. In 5 of the 6 Epstein-Barr virus-negative BL cell lines examined, the cells constitutively express undetectable or low levels of Fas and are resistant to Fas-mediated signals induced by monoclonal anti-Fas antibody. All 6 of the BL cell line B cells upregulate Fas in response to CD40 ligation, and in 4 of the cases they become sensitive to Fas- mediated death signals. In one BL cell line, the cells are constitutively sensitive to Fas-mediated cytolysis and are unaffected by CD40 signals. Next, we applied these immunologic manipulations to cells from a refractory clinical sample and observed that the tumor cells could be induced to express Fas and undergo apoptosis in our system. These results establish CD4+ T cells and the Fas-Fas ligand system as important immune regulators of Burkitt's lymphoma B cells and indicate that the susceptibility of tumor cells to Fas-mediated death signals can be modulated by specific activation events at the cell surface.

scholarly journals Heterogeneity of helper/inducer T lymphocytes. II. Effects of interleukin 4- and interleukin 2-producing T cell clones on resting B lymphocytes.

1988 ◽  
Vol 167 (4) ◽  
pp. 1350-1363 ◽  
Author(s):  
W H Boom ◽  
D Liano ◽  
A K Abbas
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Th1 Cells ◽  
Specific Class ◽  
Ifn Gamma ◽  
T Cell Clones ◽  
Cell Clones

To compare the helper function of murine T cell clones that secrete IL-2 and IFN-gamma (Th1 cells) or IL-4 and IL-5 (Th2), purified resting B cells were stimulated with F(ab')2 rabbit anti-mouse Ig (RAMG) and rabbit Ig-specific, class II MHC-restricted cloned T cells belonging to the two subsets. Both Th2 clones examined induced strong proliferative responses of B cells in the presence of RAMG, as well as the secretion of IgM and IgG1 antibodies. In contrast, the Th1 clones tested failed to stimulate B cell growth or antibody secretion. Th2-mediated B cell activation was dependent on IL-4 and IL-5, and was also inhibited by IFN-gamma or IFN-gamma produced by Th1 cells present in the same cultures. However, the failure of Th1 cells to help resting B cells could not be reversed with neutralizing anti-IFN-gamma antibody. In addition to this inhibitory effect, IFN-gamma was required for the secretion of IgG2a antibody, particularly when B cells were stimulated with polyclonal activators such as LPS. Finally, both sets of T cell clones secreted lymphokines when stimulated with purified B cells and RAMG. These experiments demonstrate that T cells that differ in lymphokine production also differ in their ability to help B cells as a result of cognate interactions at low concentrations of antigens. Moreover, IL-4, IL-5, and IFN-gamma serve different roles in the T cell-dependent proliferative and differentiative responses of resting B lymphocytes.

GMP-Grade Generation of B-Lymphocytes for Adoptive Immunotherapy in Patients After Allogeneic Stem Cell Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4352-4352
Author(s):  
Julia Winkler ◽  
Michael Mach ◽  
Juergen Zingsem ◽  
Volker Weisbach ◽  
Andreas Mackensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Lymphocytes ◽  
B Cell ◽  
B Lymphocytes ◽  
Clinical Setting ◽  
One Step ◽  
Enrichment Protocol ◽  
The One

Abstract Abstract 4352 Background and objectives: We have recently shown that memory B-lymphocytes from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B-cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of post-HSCT immunodeficiency (Klenovsek et al., 2007, Blood 110: 3472–9). As adoptive transfer of B-cells has not been used before in a clinical setting, it is necessary to establish a technology for the generation of GMP-grade B-cell products. Methods: Starting from the leukapheresis of healthy donors, B-cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS∧TM device. A one-step protocol was used for positive enrichment of B-lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T-lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads. Results: The leukapheresis contained a mean of 9.0×10∧8 CD19-positive B-cells (4.5–12.4 ×10∧8). After the one-step positive purification strategy a mean purity of CD20∧+ B-lymphocytes of 78.1% with a recovery of 32–41% was obtained. With the two-step T-cell depletion/B-cell enrichment protocol we achieved a mean purity of 96.4 % (93.4–97.8%) with a slightly lower recovery of 14–37%. The absolute B-cell numbers obtained in the product were 1.3 to 4.0 ×10∧8 and 1.7 to 2.6 ×10∧8 for the one-step positive enrichment and the two-step protocol, respectively. Importantly, the absolute number of T-cells was lower in cell products after the two-step protocol (0.1 to 0.9 ×10∧6 T-cells) as compared to the one-step positive CD19-enrichment (1.6 to 3.4 ×10∧6 T-cells). Assuming a patient with 70 kg body weight, the B-cell products obtained after the combined CD3-depletion and CD19-enrichment contained less then 4×10∧4 T-lymphocytes/kg bodyweight, which is a critical threshold number of T-cells in haploidentical HSCT. The B-cell products showed antibody production after in vitro stimulation in a limiting dilution assay and showed excellent viability after cryopreservation. Conclusions: A GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS∧TM technology. (Supported by BayImmuNet) Disclosures: No relevant conflicts of interest to declare.

scholarly journals CELL INTERACTIONS IN THE IMMUNE RESPONSE IN VITRO

1972 ◽  
Vol 136 (4) ◽  
pp. 737-760 ◽  
Author(s):  
Marc Feldmann
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Interaction ◽  
T And B Lymphocytes ◽  
Activated T Cells ◽  
Cell Cooperation

The mechanism of interaction of T and B lymphocytes was investigated in an in vitro hapten carrier system using culture chambers with two compartments separated by a cell impermeable nucleopore membrane. Because specific cell interaction occurred efficiently across this membrane, contact of T and B lymphocytes was not essential for cooperation which must have been mediated by a subcellular component or "factor." By using different lymphoid cell populations in the lower culture chamber and activated thymus cells in the upper chamber (with antigen present in both), it was found that the antigen-specific mediator acted indirectly on B cells, through the agency of macrophages. Macrophages which had been cultured in the presence of activated T cells and antigen acquired the capacity to specifically induce antibody responses in B cell-containing lymphoid populations. Trypsinization of these macrophages inhibited their capacity to induce immune responses, indicating that the mediator of cell cooperation is membrane bound. By using antisera to both the haptenic and carrier determinants of the antigen as blocking reagents, it was demonstrated that the whole antigen molecule was present on the surface of macrophages which had been exposed to activated T cells and antigen. Because specifically activated T cells were essential a component of the antigen-specific mediator must be derived from these cells. By using anti-immunoglobulin sera as inhibitors of the binding of the mediator to macrophages, the T cell component was indeed found to contain both κ- and µ-chains and was thus presumably a T cell-derived immunoglobulin. It was proposed that cell cooperation is mediated by complexes of T cell IgM and antigen, bound to the surface of macrophage-like cells, forming a lattice of appropriately spaced antigenic determinants. B cells become immunized by interacting with this surface. With this mechanism of cell cooperation, the actual pattern of antigen-B cell receptor interactions in immunization would be the same with both thymus-dependent and independent antigens. An essential feature of the proposed mechanism of cell cooperation is that macrophage-B cell interaction must occur at an early stage of the antibody response, a concept which is supported by many lines of evidence. Furthermore this mechanism of cell interaction can be elaborated to explain certain phenomena such as the highly immunogenic macrophage-bound antigen, antigenic competition, the distinction between immunity and tolerance in B lymphocytes, and the possible mediation of tolerance by T lymphocytes.

Rituximab during Pregnancy: Serum Levels, B Cell Counts and Immunoglobulin Levels in Mother and Child.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1508-1508
Author(s):  
Birte Friedrichs ◽  
Markus Tiemann ◽  
Michael K. Wenger ◽  
Karl Verpoort ◽  
Norbert Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Serum Levels ◽  
Burkitt’S Lymphoma ◽  
Cell Counts ◽  
Mother And Child ◽  
Newborn Child ◽  
Immunoglobulin Levels

Abstract Recently the first cases of lymphoma patients treated with rituximab and combination chemotherapy during pregnancy were reported. We report on a patient with Burkitt’s lymphoma who was treated with rituximab and CHOP therapy early during pregnancy. We were able to monitor rituximab concentrations, B-/T- cell counts and immunoglobulin levels. After delivery these parameters were also measured in the newborn child. A 35-year-old female was diagnosed with CD20+ Burkitt’s lymphoma of the left breast in week 15 of pregnancy. The minimum stage was IIEA; however, the patient also had hepatosplenomegaly. We started treatment with four weekly infusions of rituximab (375mg/m2)(week 16,17,18,19). Treatment was well tolerated with minimal side effects; a minor response was documented by MRI of the breast and ultrasound of previously enlarged axillary lymph nodes. At that time, a decision was made to continue treatment with 6 courses of Cyclophosphamid, Doxorubicin, Vincristin and Prednison (CHOP) at 3 week intervals (week 21, 24, 27, 30, 33, 36). The first 4 courses were preceded by rituximab (375mg/m2). Immunotherapy was tolerated without significant problems. At the end of therapy (week 37) a complete remission was achieved, again documented by MRI and ultrasound. During therapy the child’s growth and intrauterine development were closely monitored by the attending gynecologist. No deviation from normal development were registered. In week 41 of pregnancy the patient delivered a healthy girl (3780g, 55cm, APGAR score 9/10/10) via caesarean section. The girl is now 19 month old, has repeatedly been seen by her pediatrician who reported completely normal growth and developmental status. The mother received high-dose therapy (BEAM) followed by autologous peripheral blood stem cell transplantion 2 months after delivery and has remained in CR with a normal performance status. As the mother has been extremely compliant we were able to repeatedly measure B cell counts, immunoglobulin levels and rituximab concentrations not only in the patient but also in the baby (table 1). Interestingly, at the time of birth very high serum levels of rituximab were measured in the child. Nonetheless a normal B cell recovery was seen during the following weeks, immunological status reached normal values 4 month after delivery and no overt infectious complications have been reported. As to our knowledge, for the first time data of rituximab serum concentrations are available from mother and child. To conclude, in this case combination of immuno- and chemotherapy could be safely administered, achieving a CR in the patient without causing any mental or developmental retardation in the newborn child. In accordance with other reports, this case supports the safety and efficacy of Rituximab administration during pregnancy. Table 1: B/T cell counts and Rituximab concentrations in mother and child during and after treatment with R-CHOP Time Mother Child *values within normal range week of pregnancy/ B-cells T-cells Rituximab B-cells T-cells Rituximab week after delivery CD 19+ CD 3+ CD 19+ CD 3+ cells/μl cells/μl ng/mL cells/μl cells/μl ng/mL 20 0 946 27 0 640 34 4 337 at birth 0 779 9750 0 93 32095 +4 0 759 70 6616* 5399 +18 37 504 <500 1460* 5475* 700

scholarly journals Isolation and immunological characterization of a major surface glycoprotein (gp54) preferentially expressed on certain human B cells.

1979 ◽  
Vol 149 (6) ◽  
pp. 1424-1437 ◽  
Author(s):  
C Y Wang ◽  
S M Fu ◽  
H G Kunkel
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Cytotoxicity Assay ◽  
Leukemic Cells ◽  
Immunofluorescent Staining ◽  
Single Individual

A major membrane glycoprotein with mol wt of approximately 54,000 has been isolated from membrane preparations of B-type lymphoid cell lines. Antiserum prepared against the isolated material specifically precipitated this glycoprotein from membranes labeled by surface radioiodination or by metabolic labeling. This antiserum was shown by complement-mediated cytotoxicity assay, membrane immunofluorescent staining, and by quantitative absorption analysis to react preferentially with certain B-lymphoblastoid cell lines, with a minor population of peripheral blood B lymphocytes, and a major population of tonsillar B lymphocytes. Certain B-cell leukemias also expressed the antigen, whereas others did not. Considerable variability was observed among positive B cells in the intensity of fluorescent staining even among the leukemic cells from a single individual. Although T cells, including T cells, were negative by direct immunofluorescent and cytotoxicity assay, evidence for low levels of the antigen on the cells of T cell lines was obtained. The whole specific antiserum and its F(ab')2 fragments stimulated B lymphocytes to proliferate. This proliferation did not produce differentiation to plasma cells and was T-cell independent. The monovalent Fab fragments had no effect. None of these preparations timulated T cells. The possibility that this antigen, termed gp54, may play some role in B-cell activation is discussed.

scholarly journals Human Non-Germinal Center B Cell Interleukin (IL)-12 Production Is Primarily Regulated by T Cell Signals CD40 Ligand, Interferon γ, and IL-10: Role of B Cells in the Maintenance of  T Cell Responses

1999 ◽  
Vol 189 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Joachim L. Schultze ◽  
Sabine Michalak ◽  
Joel Lowne ◽  
Adam Wong ◽  
Maria H. Gilleece ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Synergistic Effects ◽  
Cell Receptor ◽  
Cd40 Ligation ◽  
Cd40 Activation ◽  
Ifn Γ

Interleukin (IL)-12 is expressed mainly in antigen-presenting cells after challenge with microbial material or after CD40 activation. Although IL-12 was cloned from human Epstein-Barr virus (EBV)-transformed B cell lines, surprisingly, CD40 ligation on murine B cells did not lead to IL-12 production, suggesting that murine B cells do not produce IL-12. Here we demonstrate that a subset of human tonsillar B cells can be induced to express and secrete bioactive IL-12. The major stimulus to produce IL-12 in human B cells was CD40 ligation. In contrast, B cell receptor cross-linking did not induce IL-12. Expression of IL-12 after CD40 activation was restricted to CD38−IgD± non-germinal center (non-GC) B cells. CD40 ligation and interferon (IFN)-γ exhibited synergistic effects on IL-12 production, whereas IL-10 abrogated and IL-4 significantly inhibited IL-12 production by these B cells. In contrast to IL-12, production of IL-6 is conversely regulated, leading to significant increase after CD40 ligation in the presence of the T helper type 2 (Th2) cytokine IL-4. Cord blood T cells skewed towards either a Th1 or a Th2 phenotype maintained their cytokine expression pattern when restimulated with allogeneic resting B cells. Blockade of CD40 and/or IL-12 during T–B interaction significantly reduced IFN-γ production by the T cells. This suggests a model whereby B cells produce either IL-12 or IL-6 after contact with T cells previously differentiated towards Th1 or Th2. Furthermore, IL-12 and IL-6 might provide a positive feedback during cognate T–B interactions, thereby maintaining T cells' differentiation pattern during amplification of the immune response.

scholarly journals Current Clinical Applications and Future Perspectives of Immune Checkpoint Inhibitors in Non-Hodgkin Lymphoma

2020 ◽  
Vol 2020 ◽  
pp. 1-18
Author(s):  
John Apostolidis ◽  
Ayman Sayyed ◽  
Mohammed Darweesh ◽  
Panayotis Kaloyannidis ◽  
Hani Al Hashmi
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Programmed Cell Death ◽  
B Cell ◽  
Cancer Cells ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors

Cancer cells escape immune recognition by exploiting the programmed cell-death protein 1 (PD-1)/programmed cell-death 1 ligand 1 (PD-L1) immune checkpoint axis. Immune checkpoint inhibitors that target PD-1/PD-L1 unleash the properties of effector T cells that are licensed to kill cancer cells. Immune checkpoint blockade has dramatically changed the treatment landscape of many cancers. Following the cancer paradigm, preliminary results of clinical trials in lymphoma have demonstrated that immune checkpoint inhibitors induce remarkable responses in specific subtypes, most notably classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma, while in other subtypes, the results vary considerably, from promising to disappointing. Lymphomas that respond to immune checkpoint inhibitors tend to exhibit tumor cells that reside in a T-cell-rich immune microenvironment and display constitutive transcriptional upregulation of genes that facilitate innate immune resistance, such as structural variations of the PD-L1 locus, collectively referred to as T-cell-inflamed lymphomas, while those lacking such characteristics are referred to as noninflamed lymphomas. This distinction is not necessarily a sine qua non of response to immune checkpoint inhibitors, but rather a framework to move the field forward with a more rational approach. In this article, we provide insights on our current understanding of the biological mechanisms of immune checkpoint evasion in specific subtypes of B-cell and T-cell non-Hodgkin lymphomas and summarize the clinical experience of using inhibitors that target immune checkpoints in these subtypes. We also discuss the phenomenon of hyperprogression in T-cell lymphomas, related to the use of such inhibitors when T cells themselves are the target cells, and consider future approaches to refine clinical trials with immune checkpoint inhibitors in non-Hodgkin lymphomas.

scholarly journals COMPLEMENT-DEPENDENT B-CELL ACTIVATION BY COBRA VENOM FACTOR AND OTHER MITOGENS?

1974 ◽  
Vol 139 (2) ◽  
pp. 337-354 ◽  
Author(s):  
Peter Dukor ◽  
Gebhard Schumann ◽  
Roland H. Gisler ◽  
Manfred Dierich ◽  
Wolfgang König ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Cultures ◽  
Antibody Formation ◽  
Cell Activation ◽  
B Cell Activation

It has been proposed that two distinct signals are required for the triggering of the precursors of antibody-forming bone marrow-derived cells (B cells): (a) the binding of antigen or of a mitogen to the corresponding receptor sites on B-cell membranes and (b) the interaction of activated C3 with the C3 receptor of B lymphocytes. There is growing evidence that B-cell mitogens and T (thymus-derived cell)-independent antigens are capable of activating the alternate pathway of the complement system (bypass). Therefore, the effect of another potent bypass inducer was investigated with regard to B-cell activation and the role of C3. Purified, pyrogen-free cobra venom factor was mitogenic for both T and B lymphocytes (cortisone-resistant mouse thymus cells and lymph node lymphocytes from congenitally athymic mice). Venom factor could substitute for T cells by restoring the potential of antibody formation to sheep red blood cells in mouse B-cell cultures supplemented with macrophages or 2-mercaptoethanol. Venom factor may be capable of conferring activated C3 to the C3 receptor of B lymphocytes: preincubation of lymphoid cells with homologous serum or plasma, 10 mM EDTA, and sepharose-coupled venom factor converted with serum to an enzyme active against C3, inhibited their capacity to subsequently form rosettes with sheep erythrocytes sensitized with amboceptor and C5-deficient mouse complement. In the absence of EDTA, preincubation of freshly prepared B-cell suspensions with C3-sufficient homologous serum also blocked their subsequent interaction with complement-sensitized erythrocytes and at the same time rendered them reactive to an otherwise T-cell-specific mitogen. Moreover, mitogen induced B-cell proliferation in lymph node (but not in spleen) cell cultures, appeared to depend on the availability of exogenous C3: zymosan-absorbed fetal bovine serum (only 8.3% site-forming units remaining) supported T-cell activation by phytohemagglutinin, concanavalin A, and venom factor, but failed to sustain B-cell stimulation by pokeweed mitogen, lipopolysaccharide, and venom factor. T-cell-dependent antibody formation in composite cultures containing T cells or T-cell-substituting B-cell mitogens, B cells, and macrophages, always required the presence of C3-sufficient serum.

A “Survival of the Fittest” Mechanism for Weeding Out Potentially Lymphomagenic B-Cells during Germinal Center B-Cell Differentiation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 559-559
Author(s):  
Weimin Ci ◽  
Jose M. Polo ◽  
Stella M. Ranuncolo ◽  
Ari Melnick
Keyword(s):  
Dna Damage ◽  
Quality Control ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Affinity Maturation ◽  
Mrna Levels

Abstract During normal T-cell dependent immune responses, activated B-cells differentiate into germinal center (GC) centroblasts, which tolerate simultaneous genomic recombination and rapid clonal expansion in order to produce high affinity antibodies. Upregulation of the BCL6 transcriptional repressor is required for centroblasts to acquire this phenotype, since it can repress DNA damage sensing and checkpoint genes. Genetic lesions that cause constitutive expression of BCL6 are common oncogenic events in human diffuse large B-cell lymphomas (DLBCL) and presumably contribute to malignant transformation by sustaining the centroblast phase and facilitating accumulation of genetic errors. We hypothesized that centroblasts must have evolved a mechanism to rapidly terminate the genomic instability phenotype in order to limit the likelihood of malignant transformation. A recent report (Allen et. al. PMID: 17185562) showed that centroblasts and GC T-cells make direct physical contact for ∼30 minutes during affinity maturation. We wondered whether CD40 signaling from T-cells could disrupt the function of BCL6 within this timeframe. Accordingly, we found in ChIP assays that CD40 signaling in B-cells can disrupt the ability of BCL6 to recruit the SMRT and N-CoR corepressors within minutes, at which time RNA polymerase II moves from the promoter to the exons of BCL6 target genes, histones became acetylated, and mRNA levels increase. We showed that signaling from CD40 to N-CoR was dependent on NFkB, while signaling to SMRT appeared to be related to MAP kinase phosphorylation cascades. Two BCL6 targets regulated in this manner are ATR and p53. Accordingly, although CD40 can promote survival of intact GC B-cells, CD40 induced cell death of centroblasts in the presence of higher levels of DNA damage led to cell death, rather than survival. Washout of CD40 after 60 minutes to emulate transient T-cell contact permitted BCL6 target gene mRNA levels to return to their repressed levels, demonstrating that this is a reversible process, which could allow centroblasts that pass quality control to either continue proliferation or undergo terminal differentiation. In order to identify direct targets of BCL6 subject to this regulatory mechanism, we performed ChIP-on-chip of BCL6 in primary human tonsilar centroblasts on a 24,000 promoter array. 915 genes were identified with a cut-off of 99.9th percentile (FDR<0.026), while 1880 genes were identified with a cutoff at the 99th percentile (FDR<0.12). These natural BCL6 targets are functionally related to DNA damage response, transcriptional repression, protein translation, NFkB signaling and others, re-expression of which may play a critical role in facilitating quality control of centroblasts during affinity maturation. Taken together, these data suggest that transient CD40 signaling in the GC might allow T-cells to “weed out” heavily damaged centroblasts while at the same time promoting survival of only the “fittest” B-cells, which could undergo differentiation or additional rounds of proliferation. Others have shown that sustained CD40 signaling can downregulate BCL6 at longer timepoints, possibly reflecting longer exposure of B-cells to this signaling pathway that might occur towards the end of their cycle through the germinal center. Therefore, CD40 can inhibit BCL6 through two different mechanisms, each with potentially different functions during B-cell maturation. Loss of either mechanism is likely to contribute to lymphomagenesis.

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