Germinal Center Cells Research Articles

Rb Protects B-Lineage Hematopoietic Progenitor Cells From Oxidative Stress and Exhaustion

Abstract 1315 Background:Multiple myeloma (MM) is the second most common hematologic malignancy in the United States. Chromosome 13 deletions are found in approximately one half (50%) of all patient samples and is associated with worse outcomes. The retinoblastoma gene (Rb1) has been implicated as a candidate tumor suppressor at 13q14, however, unmutated Rb1 remains expressed from retained alleles in the majority of cases. We sought to explore the role of Rb1 in plasma cells by generating mice with deficiency of Rb1 targeted to late B-cell development. Methods:The Rb family member p107 can compensate for Rb1 function, so we crossed Rb1Flox/Flox and P107−/−mouse strains with Cγ1-Cre knock-in mice that express the Cre recombinase specifically in germinal center (GC) B-cells to generate mice with tissue-specific deletion of Rb in germinal center cells (Cg1-Rb-KO), and we aged these mice to assess disease development. Results:In Cg1-Rb-KO mice, we demonstrated the Rb1 locus was successfully recombined in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). Recombination was also detectable in bone marrow B-cell but not myeloid cell progenitors. Cg1-Rb-KO mice developed normally but became ill starting at 40 weeks of age. Sick mice demonstrated weight loss, hypoplastic spleens, osteopenia and significant lymphopenia. Thorough analysis of bone marrow and spleen hematopoietic progenitor cell subsets by multi-parameter flow cytometry at the time of death revealed dramatic reductions in marrow B-cell (Hardy fractions C-D, from 3.18×103to 40 cells and from 1.4×106 to 7.5×104cells respectively), suggesting an effective collapse of the B-cell lineage in mice lacking Rb. Examination of Cg1-Rb-KO mice prior to becoming sick (age 30–35 weeks) demonstrated increased numbers of B-cell progenitors in the bone marrow, consistent with Rb’s known role in cell cycle regulation. Together, our results suggest Cg1-Rb-KO mice develop benign B-cell hyperplasia followed by a lethal collapse of the B-cell lineage. To help explain this B-cell exhaustion phenotype, we measured reactive oxygen species (ROS) in the B-cell of young Cg1-Rb-KO mice. We observed significantly increased ROS in Cg1-Rb-KO mice at baseline and following LPS stimulation ex vivo and in vivo compared to p107-KO control mice, suggesting that B-cells from Cg1-Rb-KO mice have elevated levels of oxidative stress. Conclusion:Rb1 is important for maintenance of B-cell homeostasis and protects B-lineage progenitor cells from oxidative stress and exhaustion. Rb1 deletion was not sufficient for B-cell transformation and additional mutations are likely required for MM development in the context of complete RB1 loss. Disclosures:Vij:Millennium: Speakers Bureau.

The Transcriptional Profile of Follicular Lymphoma Associated Regulatory T-Cells (Tregs) Is More Consistent with Tregs Having a Germinal Center Localization and the Ability to Suppress Follicular Helper T-Cells

AbstractAbstract 1536Recent studies have suggested that regulatory T-cells (Tregs) play a critical role in the pathogenesis of follicular lymphoma (FL). We have previously shown that the Tregs infiltrating FL lymph nodes (FLN Tregs) are quantitatively and qualitatively different than those infiltrating normal and reactive nodes (NLN, RLN, respectively). To gain insight into how such Treg populations differ, and how FLN Tregs may impact FL progression and response to therapy, we performed RNA sequence analyses on flow sorted Tregs (CD3+CD4+CD25brightCD127−) from NLN (n=10), FLN (n=12) and RLN (n=7).Using the RefSeq annotated set of mRNA we first identified 11686 annotated genes that were expressed in each of the Treg groups. Using pairwise unequal variance t-tests at p<0.01, we identified 798 genes that differed between NLN and FLN, 589 that differed between NLN and RLN, and 475 genes that differed between FLN and RLN. The transcriptome profile of the FLN Tregs differed most from that of NLN Tregs, with 281 genes increased in FLN relative to NLN, and 517 genes decreased in FLN Tregs relative to NLN Tregs. In contrast, 404 genes were increased and 70 genes decreased in RLN Tregs relative to FLN Tregs suggesting that differences in the transcriptome profile between NLN and FLN Tregs are due, in part, to inflammatory signals shared between FLN and RLN. Tregs, which express FOXP3, are a heterogeneous population which share a “core suppressive module” of CD25 and CTLA-4, but which adapt to microenvironmental signals by co-opting/or up-regulating the canonical transcription factor (TF) of the T-cell population the Treg needs to suppress. This results in modulation of the Treg chemokine receptors in such a fashion that allows these Tregs to traffic to and suppress the specific T-cell population defined by their TF expression. Relative to NLN, FLN Tregs have similar expression of FOXP3 and CD25 but greater expression of CTLA-4 (confirmed at the protein level), which may account, in part, for the greater suppressive activity of FLN Tregs compared to NLN Tregs. Expression of other genes that differ between NLN and FLN which encode for proteins that modulate Treg suppression include IL-10 (up in FLN relative to NLN), S1PR1 and ENTPD1 (both down in FLN relative to NLN), which all favor greater suppression by FLN Tregs. In contrast LGALS1 and NT5E expression (both down in FLN relative to NLN) would favor less suppression by FLN Tregs. Relative to NLN, FLN Tregs have greater BCL-6 expression, consistent with a population that suppress follicular helper T-cells (Tfh), which support normal germinal center (GC) and FL-B cell survival. Consistent with their increased expression of BCL-6, FLN Tregs have greater expression of CXCR5, CXCR4 and CXCL13 and decreased expression of CCR7 compared to that of NLN Tregs, a repertoire associated with GC trafficking. The significant lower expression of both S1PR1 and SELL (CD62L; p<0.000002, and fold changes of −9.0 and −2.8, respectively) that is found in FLN Tregs compared to NLN Tregs is consistent with the greater nodal retention. The lower expression of RORc found in the FLN Tregs would predict for decreased IL-17 secretion and their elevated levels of STAT3 which would support the ability of the FLN Tregs to inhibit Th17 cells.In conclusion, the comparison of the transcriptome profiles of FLN and NLN Tregs; a) suggest that the observed differences are shaped, in part, by inflammatory signals also present in RLN; b) lay the foundation of a hypothesis to explain the molecular basis for their greater immunosuppressive capacity; and c) suggest that FL Tregs are ‘poised' to localize in the GC, be strongly retained in the lymph node and have enhanced potential to suppress Tfh and Th17 cells. Such findings offer new insights into how FL Tregs may contribute to the biology of these B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

MEF2B Mutations Lead to De-Regulated Expression of the BCL6 Oncogene in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma

Abstract 1284Diffuse large B-cell lymphoma (DLBCL) and Follicular Lymphoma (FL) are the most common forms of non-Hodgkin’s lymphoma in the adult, accounting for approximately 75% of lymphoma diagnoses. Recent technological advances, including whole-genome DNA and RNA sequencing and gene copy-number analysis, have provided a comprehensive view of the genomic landscape of DLBCL, allowing new insights in the somatic genetic lesions that are associated with the pathogenesis of this malignancy. Among the genetic alterations that are recurrently found in DLBCL and FL, but remain of unclear functional significance, are the mutations involving the MEF2B gene. MEF2B is a member of the myocyte enhancer-binding factor 2 (MEF2) family of transcription factors whose activity is dependent on association with specific co-repressors (including CABIN1 and HDACs) and co-activators in response to multiple signaling pathways. Overall, ∼11% of DLBCL and ∼12% of FL cases reported carry mutations in MEF2B (Morin Nature 2011; Pasqualucci Nat Genet 2011; Lohr PNAS 2012).We showed that within the mature B-cell lineage, MEF2B expression is restricted to germinal center (GC) B-cells. The analysis of the B-cell interactome, a network of protein-protein and protein-DNA interactions generated by reverse-engineering a large dataset of B-cell phenotypes, showed that MEF2B was uniquely connected to BCL6, a proto-oncogene and well-characterized master regulator of the GC reaction. We demonstrated that MEF2B directly binds to the promoter of BCL6 and leads to its trans-activation in GC B-cells. Consistently, silencing of MEF2B in GC-derived lymphoma cell lines led to BCL6 down-regulation and impairment of cell cycle progression and proliferation, suggesting that normal and malignant GC cells are dependent on MEF2B expression.Approximately 80% of the DLBCL and FL mutated cases carry missense mutations clustered in the N-terminal conserved MADS-box and MEF2 functional domains, suggesting that they may have a relevant impact on MEF2B function. In a second group of cases (∼20%), mutations affect the C-terminal half of the MEF2B protein, and are mostly represented by frameshift and nonsense mutations, which truncate or modify the C-terminus of the protein.In order to functionally characterize these mutations, we first investigated whether DLBCL- and FL-associated MEF2B mutations affected the ability to regulate the transcription of BCL6. Using a reporter construct containing the native BCL6 promoter region responsive to MEF2B, we demonstrated that most of the N-terminal mutations cause increased transcriptional activity as tested on the BCL6 promoter. The analysis of the N-terminal MEF2B crystal structure, upon mapping the mutated residues, predicted that these mutations may interfere with the ability of MEF2B to heterodimerize with the CABIN1 co-repressor. Indeed, we showed that these MEF2B mutant proteins fail to bind CABIN1 and are resistant to its transrepressive activity. Conversely, C-terminal MEF2B mutations lead to truncated MEF2B proteins lacking the domains responsive to two independent post-transcriptional modifications, namely PKA-mediated phosphorylation and sumoylation. We showed that MEF2B is in fact phosphorylated by PKA and sumoylated in vivo, that both of these modifications lead to negative regulation of MEF2B transcriptional activity, and that lymphoma-associated C-terminal mutants fail to be negatively regulated by PKA-mediated phosphorylation and sumoylation.In summary, these results identify MEF2B as an upstream regulator of BCL6 and GC formation, which is required for lymphoma proliferation. Lymphoma-associated MEF2B mutations may contribute to lymphomagenesis, at least in part, by deregulating the expression of the BCL6 oncogene. Thus, targeting MEF2B may represent an alternative therapeutic approach to block BCL6 and cell proliferation in DLBCL and FL. Disclosures:No relevant conflicts of interest to declare.

Lyn Signaling To Upregulate GANP Is Critical for the Survival of High-Affinity B Cells in Germinal Centers of Lymphoid Organs

Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn(-/-)) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn(-/-) mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenyl-chicken γ-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp(33)-to-Leu mutation in the IgV(H)-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs.

Follicular helper T cells in immune homeostasis

Follicular helper T cells in immune homeostasis

ER stress in diffuse large B cell lymphoma: GRP94 is a possible biomarker in germinal center versus activated B-cell type

The process of B-cell development is characterized by the activation of the unfolded protein response. Under certain circumstances, the unfolded protein response can be manipulated in a cell death-inducing way. Therefore, tackling the unfolded protein response might be an attractive strategy in the treatment of diffuse large B-cell lymphomas.Our research showed more basal unfolded protein response activity and differences in the inducibility of ER stress in activated B-cell versus germinal center cell lines. Moreover, the diffuse large B cell lymphoma patient data revealed that the glucose-regulated protein 94 is new potential discriminator for diffuse large B cell lymphoma.

Alteration of the germinal center checkpoint by the lupus associated NZM2410/NZW SLAM family genes in the Sle1b sub-locus (171.40)

Abstract C57BL/6 (B6) mice carrying the Sle1b sub-locus that harbors the lupus-associated NZM2410/NZW SLAM family genes produce anti-nuclear antibodies (ANA). However, the role and mechanisms of alteration in the germinal center (GC) checkpoint in the development of autoantibodies in these mice (named B6.Sle1b mice) is not clear. Here we show significantly higher spontaneously formed GC (Spt-GC) response in aged B6.Sle1b mice compared to B6 controls. The increased Spt-GC response in B6.Sle1b mice did not result from a generalized defect in the response of B cells expressing Sle1b as no difference was observed between two groups of mice in the anti-sheep red blood cell (sRBC) GC responses. We found a significant increase in CD4+CXCR5hiPD-1hi follicular helper T cells (TFH) within Spt-GC and anti-sRBC GCs in B6.Sle1b mice. The increase in Spt-GC and TFH cell responses in B6.Sle1b mice was not due to reduced CD4+CD25+Foxp3+ T effector (iTreg) and TFH regulatory (TFR) cells in GCs. Sle1b GCs compared to controls had significantly higher proliferating B cells. This elevated Spt-GC response in B6.Sle1b mice was associated with increased DNA and nucleosome-specific plasma cells (PCs), memory B cells and ANAs. These data indicate that the mechanisms that control the formation of Spt-GCs and generation of autoreactive B and TFH cells in GCs are altered by the NZM2410/NZW SLAM family genes located in Sle1b.

BTB-POZ transcription factors recruit the E3 ubiquitin ligase Cullin 3 to initiate lymphoid effector programs (111.38)

Abstract The differentiation of several T and B cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodeling. We report that PLZF, the BTB-POZ transcription factor directing the innate-like effector program of NKT thymocytes was prominently associated with cullin 3 (Cul3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF induced the transport of Cul3 from the cytosol to the nucleus where the two proteins were associated within a chromatin associated/modifier protein complex. Furthermore, PLZF expression in thymocytes resulted in selective changes of ubiquitination of multiple components of this complex. Cul3 was also found associated with another BTB-POZ transcription factor, Bcl6, which directs the B cell germinal center program. Conditional deletion in mice demonstrated an absolute, cell-intrinsic requirement of Cul3 for the development of NKT cells and germinal center B cell responses. We conclude that distinct lineage-specific BTB-POZ transcription factors recruit Cul3 to alter the ubiquitination pattern of their associated chromatin associated/modifier complex. We propose that this novel function is essential to direct the differentiation of several T and B lymphocyte effector programs, and may also be involved in the oncogenic role of PLZF and Bcl6 in leukemias and lymphomas.

Germinal centers

Germinal centers

Spatial Alterations between CD4+ T Follicular Helper, B, and CD8+ T Cells during Simian Immunodeficiency Virus Infection: T/B Cell Homeostasis, Activation, and Potential Mechanism for Viral Escape

HIV/SIV infections induce chronic immune activation with remodeling of lymphoid architecture and hypergammaglobulinemia, although the mechanisms leading to such symptoms remain to be fully elucidated. Moreover, lymph nodes have been highlighted as a predilection site for SIV escape in vivo. Following 20 rhesus macaques infected with SIVmac239 as they progress from pre-infection to acute and chronic infection, we document for the first time, to our knowledge, the local dynamics of T follicular helper (T(FH)) cells and B cells in situ. Progression of SIV infection was accompanied by increased numbers of well-delineated follicles containing germinal centers (GCs) and T(FH) cells with a progressive increase in the density of programmed death-1 (PD-1) expression in lymph nodes. The rise in PD-1(+) T(FH) cells was followed by a substantial accumulation of Ki67(+) B cells within GCs. However, unlike in blood, major increases in the frequency of CD27(+) memory B cells were observed in lymph nodes, indicating increased turnover of these cells, correlated with increases in total and SIV specific Ab levels. Of importance, compared with T cell zones, GCs seemed to exclude CD8(+) T cells while harboring increasing numbers of CD4(+) T cells, many of which are positive for SIVgag, providing an environment particularly beneficial for virus replication and reservoirs. Our data highlight for the first time, to our knowledge, important spatial interactions of GC cell subsets during SIV infection, the capacity of lymphoid tissues to maintain stable relative levels of circulating B cell subsets, and a potential mechanism for viral reservoirs within GCs during SIV infection.

Promotion of a Functional B Cell Germinal Center Response after Leishmania Species Co-Infection Is Associated with Lesion Resolution

Co-infection of C3HeB/FeJ (C3H) mice with both Leishmania major and Leishmania amazonensis leads to a healed footpad lesion, whereas co-infection of C57BL/6 (B6) mice leads to non-healing lesions. This inability to heal corresponds to a deficiency in B cell stimulation of the macrophage-mediated killing of L. amazonensis in vitro and a less robust antibody response. The mechanism that leads to healing of these lesions is not completely known, although our studies implicate the B cell response as having an important effector function in killing L. amazonensis. To understand more completely this disparate clinical outcome to the same infection, we analyzed the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There were more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells, and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks postinfection. Interleukin (IL)-21 production and IL-21 receptor expression in both mouse strains, however, were similar at 2 weeks, suggesting that the difference in the anti-Leishmania response in these mouse strains may be due to differences in T follicular cell commitment or intrinsic B cell differences. These data support the idea that functional B cells are important for healing L. amazonensis in this infectious disease model.

Tracking Murine Gammaherpesvirus 68 Infection of Germinal Center B Cells In Vivo

Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency – which should facilitate studies to track MHV68 latently infected cells at late times post-infection.

Fluorescent In Vivo Detection Reveals that IgE+ B Cells Are Restrained by an Intrinsic Cell Fate Predisposition

IgE antibodies may be protective in parasite immunity, but their aberrant production can lead to allergic disease and life-threatening anaphylaxis. Despite theimportance of IgE regulation, few studies have directly examined the B cells that express IgE, because these cells are rare and difficult to detect. Here, we describe fluorescent IgE reporter mice and validate a flow cytometry procedure to allow sensitive and specific identification of IgE-expressing B cells invivo. Similar to IgG1(+) cells, IgE(+) B cells differentiated into germinal center (GC) B cells and plasma cells (PCs) during primary immune responses toa Tcell-dependent hapten-protein conjugate and the helminth Nippostrongylus brasiliensis. However, the participation of IgE(+) B cells in GCs was transient. IgE(+) B cells had an atypical propensity to upregulate the transcription factor Blimp-1 and undergo PC differentiation. Most IgE(+) PCs were short lived andshowed reduced affinity maturation, revealing intrinsic mechanisms that restrict the IgE antibody response.

A germinal center–independent pathway generates unswitched memory B cells early in the primary response

Memory B cells can be produced from the classical germinal center (GC) pathway or a less understood GC-independent route. We used antigen-based cell enrichment to assess the relative contributions of these pathways to the polyclonal memory B cell pool. We identified a CD38(+) GL7(+) B cell precursor population that differentiated directly into IgM(+) or isotype-switched (sw) Ig(+) memory B cells in a GC-independent fashion in response to strong CD40 stimulation. Alternatively, CD38(+) GL7(+) B cell precursors had the potential to become Bcl-6(+) GC cells that then generated primarily swIg(+) memory B cells. These results demonstrate that early IgM(+) and swIg(+) memory B cells are products of a GC-independent pathway, whereas later switched Ig(+) memory B cells are products of GC cells.

The Fas/CD95 Receptor Regulates the Death of Autoreactive B Cells and the Selection of Antigen-Specific B Cells

Cell death receptors have crucial roles in the regulation of immune responses. Here we review recent in vivo data confirming that the Fas death receptor (TNFSR6) on B cells is important for the regulation of autoimmunity since the impairment of only Fas function on B cells results in uncontrolled autoantibody production and autoimmunity. Fas plays a role in the elimination of the non-specific and autoreactive B cells in germinal center, while during the selection of antigen-specific B cells different escape signals ensure the resistance to Fas-mediated apoptosis. Antigen-specific survival such as BCR or MHCII signal or coreceptors (CD19) cooperating with BCR inhibits the formation of death inducing signaling complex. Antigen-specific survival can be reinforced by antigen-independent signals of IL-4 or CD40 overproducing the anti-apoptotic members of the Bcl-2 family proteins.

The role of Peyer’s patches in synchronizing gut IgA responses

Because Peyer’s patches (PP) are the main inductive sites for gut IgA responses we have focused this review on what we know about the function of PP germinal centers (GC). The vast majority of IgA gene sequences in the gut lamina propria (LP) are heavily mutated arguing for an origin in GC. Because PP GC formation is dependent on the presence of CD4 T cells, we speculate that all IgA responses in the normal gut are directly or indirectly T cell-dependent (TD). We hypothesize that the CD4 T cell involvement in gut IgA responses against the microbiota is different from that in systemic responses since cognate T–B cell interactions appear not to be required. In the absence of cognate interactions the function of CD4 follicular helper T cells (Tfh) in PP GC is unclear. However, production of IL-21 and IL-6 is more pronounced than in peripheral lymph nodes. Importantly, we discuss how multiple PP are involved in generating specific IgA responses to TD antigens given orally. Recently we found that oral immunization with NP-hapten conjugated to cholera toxin (NP-CT) stimulated a strong highly synchronized, oligoclonal and affinity matured IgA response. This was achieved through re-utilization of GC in multiple PP as GC IgA B cells emigrated into already established GC. Clonally related B cells were present in both inductive and effector lymphoid tissues in the gut and clonal trees involving multiple PP could be constructed in individual mice. Through adoptive transfer of B1-8hi NP-specific B cells we demonstrated that GL7+ PP B cells could enter into pre-existing GC in PP, a process that was antigen-dependent but did not to require cognate Tfh interactions. Finally, we discuss the role of PP GC for the generation of memory B cells and long-lived plasma cells in the light of contrasting findings regarding IgA memory development to colonizing commensal bacteria versus that to oral immunization with enteropathogens or TD antigens.

Chemotaxis in Densely Populated Tissue Determines Germinal Center Anatomy and Cell Motility: A New Paradigm for the Development of Complex Tissues

Germinal centers (GCs) are complex dynamic structures that form within lymph nodes as an essential process in the humoral immune response. They represent a paradigm for studying the regulation of cell movement in the development of complex anatomical structures. We have developed a simulation of a modified cyclic re-entry model of GC dynamics which successfully employs chemotaxis to recapitulate the anatomy of the primary follicle and the development of a mature GC, including correctly structured mantle, dark and light zones. We then show that correct single cell movement dynamics (including persistent random walk and inter-zonal crossing) arise from this simulation as purely emergent properties. The major insight of our study is that chemotaxis can only achieve this when constrained by the known biological properties that cells are incompressible, exist in a densely packed environment, and must therefore compete for space. It is this interplay of chemotaxis and competition for limited space that generates all the complex and biologically accurate behaviors described here. Thus, from a single simple mechanism that is well documented in the biological literature, we can explain both higher level structure and single cell movement behaviors. To our knowledge this is the first GC model that is able to recapitulate both correctly detailed anatomy and single cell movement. This mechanism may have wide application for modeling other biological systems where cells undergo complex patterns of movement to produce defined anatomical structures with sharp tissue boundaries.

Therapeutic Targeting of the Bcl-6: p53 Axis with Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma,

Abstract 3733Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's Lymphoma. Despite advances in treatment, 1/3 of patients die from their disease. Gene expression profiling has delineated three subtypes with different genetic features known to be prognostic: the Activated B-cell (ABC), Germinal Center (GC), and grey zone types. For example, ABC DLBCL is addicted to NFkB over-expression. The oncogene, BCL6, encodes a transcription factor that functions as a transcriptional repressor within normal germinal center B-cells. Constitutive activation of Bcl-6 leads to GC-type DLBCL by turning off genes expressing cell cycle dependent kinase inhibitors, and essential tumor suppressor genes, like p53. There is a critical inverse relationship between Bcl-6 and p53, the functional status of which is linked to each transcription factor's degree of acetylation. Deacetylation of Bcl-6 is required for maintaining its effects as a transcriptional repressor. Conversely, acetylation of p53 is activating when class III histone deacetylases (HDAC), also known as sirtuins, are inhibited by drugs such as niacinamide. HDAC inhibitors are presently approved for T-cell lymphoma and may require the targeting of additional pathways to be effective in B-cell lymphomas. Trichostatin A and niacinamide modulate Bcl-6 in lymphoma cell lines. One therapeutic strategy that could favorably shift the relationship between oncogenes and tumor suppressors is the pharmacologic modification of Bcl-6 and p53 using HDAC inhibitors.Eight DLBCL cell lines were screened (4 ABC: Su-DHL2, HBL-1, OCI-Ly10, RIVA; 4 GC:OCI-Ly1, OCI-Ly7, Su-DHL6, Su-DHL4) with four class I/II HDAC inhibitors (romidepsin, vorinostat, panobinostat and belinostat) in combination with niacinamide (sirtuin inhibitor) at two dose levels each at three time points. Cell growth inhibition was measured by luminescence cell viability and apoptosis flow cytometry assays. Synergy was measured by the relative risk ratio (RRR) calculation where values <1 represent synergy. Synergy was achieved in significantly greater number and intensity in the GC versus ABC cell lines. Specifically, romidepsin in combination with niacinamide achieved the greatest synergy. To analyze mechanism of action, DLBCL cell lines were treated with combinations of class I/II HDAC inhibitors and niacinamide. Cells of both GC and ABC subtypes treated with the combination resulted in increased acetylation of p53, and increased p21 and BLIMP-1 content compared to controls. These results did not correlate with cytotoxicity as the ABC cell lines did not achieve the same synergy as the GC cells. GC cells treated with the same combinations resulted in acetylation of Bcl-6 compared with controls as measured by immunoprecipitation and Western blotting assays; ABC cells do not express Bcl-6. This finding correlated with cytotoxicity implying that a rational second pathway must be targeted to shift the balance between oncogene and tumor suppressor activity to achieve effective cell kill. p300 content was also increased suggesting that treatment with HDAC inhibitors recruit or upregulate its production and activity leading to increased acetylation. Using a novel double transgenic mouse model of aggressive spontaneous B-cell lymphoma (l-myc overexpressing crossed with CD19-tagged mCherry luciferase), in vivo effects of the drug combination were studied. These mice express equal basal amounts of Bcl-6 and p53 as GC cell lines. Mice treated with niacinamide 20 mg/kg and romidepsin 2.3mg/kg IP for 5 hours achieved increased acetylation of Bcl-6 and p53, and accumulation of p21 and BLIMP1 compared with controls. Importantly, mice treated with the combination of niacinamide 40 mg/kg and romidepsin 2.3 mg/kg IP achieved decreased tumor burden as measured by bioluminescence signal intensity compared to mice treated with each drug alone and controls.Presently, we are translating these concepts and observations in a proof-of-principle phase I trial evaluating the safety of vorinostat plus niacinamide in lymphoid malignancies. By targeting the specific pathogenetic features of DLBCL, it may be possible to tailor future treatment platforms for discrete subtypes of DLBCL. [Display omitted] [Display omitted] Disclosures:Off Label Use: The drugs evaluated are not approved for use in DLBCL. O'Connor:Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

Aberrant Expression of Golgin-84 in Non-Hodgkin Lymphomas and Plasma Cell Myeloma

Abstract 4633 Background:Golgins are proteins of the Golgi complex. Several Golgins have been implicated in apoptosis. Expression of Golgin-84, a Golgin protein, is altered in apoptotic WEHI-231, a B-cell lymphoma line, suggesting that Golgin-84 may play a role in lymphoid tumorigenesis. Here, we aimed to determine the expression levels of Golgin-84 in human primary non-Hodgkin lymphomas and plasma cell myeloma. Design:Golgin-84 expression was investigated in non-Hodgkin lymphoma cell lines by using Western blot analysis and polyclonal antibodies. Using immunohistochemical stains, Western blotting analysis and Q-PCR, Golgin-84 expression was assessed in 5 reactive lymph nodes, 149 cases of primary non-Hodgkin lymphoma and 28 cases of primary plasma cell myeloma. Results:Immunohistochemical stains, Western blotting analysis and Q-PCR on 5 reactive lymph nodes demonstrated that Golgin-84 was expressed at low levels in lymphoid cells of germinal centers, mantle cells, marginal zones, and interfollicular areas. Golgin-84 was variably expressed in non-Hodgkin lymphoma cell lines tested, with the highest levels in cells from high-grade tumors (e.g. anaplastic large cell lymphoma; ALCL, Diffuse large B-cell lymphoma (DLBCL), ALCL and peripheral T-cell lymphoma unspecified (PTCL)) and the lowest levels in mantle cell lymphoma (MCL) cells. DLBCL, ALCL and PTCL frequently showed high expression of Golgin-84. Most lymphoplasmacytic lymphomas (LPL) and plasma cell myeloma (PCM) expressed high levels of Golgin-84. Expression levels of Golgin-84 were lower in MCL and low-grade B-cell non-Hodgkin lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL). Conclusions:Golgin-84 expression levels are low in lymphoid cells of normal lymph nodes. Most (>90%) cases of LPL and PCM, and at least half of cases of DLBCL, ALCL and PTCL express high levels of Golgin-84. These findings suggest that Golgin-84 may be involved in tumorigenesis or lymphoma progression, particularly in neoplasms with plasmacytic differentiation. Disclosures:No relevant conflicts of interest to declare.

Smoothened (SMO), a G-Protein-Coupled Receptor (GPCR) Activated by Hedgehog Ligands, Modulates the Activity Levels of PI3K/AKT and NF-Kβ in Diffuse Large B-Cell Lymphoma,

Abstract 3483Activated Hedgehog (HH) signaling was identified in our laboratory to contribute to cell survival, proliferation and chemotolerance of diffuse large B-cell lymphoma (DLBCL) (Leukemia 2010;24:1025 & Oncogene 2011, in press). The HH receptor complex is integrated by two main proteins, smoothened (SMO) and patched-1 (PTCH1). SMO is a seven-transmembrane G protein-coupled receptor that transduces HH signal to the cytoplasm and has glioma-associated oncogene homologue (GLI) proteins as major signaling transcription effectors. PTCH1 is a 12 transmembrane protein that inhibits SMO in the absence of HH ligands. Here, we investigated potential cross talk between SMO with the activation status of PI3K/AKT and NF-kB, two relevant oncogenic pathways in DLBCL. Using a small interfering (si)RNA approach and DLBCL cell lines of germinal center (GC) and activated B-cell (ABC) type we found that the expression levels of SMO modulate the activation status of AKT and canonical NF-KB pathways in DLBCL cells of GC type and mainly AKT in those of ABC type. In DLBCL cells of GC cell type, silencing SMO resulted in decrease of the phosphorylation levels of ser473p-AKT and ser536p-P65 and silencing PTCH1 resulted in increase of the phosphorylation levels of both proteins. The same silencing experimental approach in DLBCL cells of ABC type resulted in similar modulation in the activation status of the AKT but not, or to less extent, in the activation of NF-kB pathway. The modulation of the activation status of the NF-KB pathway was also confirmed using protein nuclear extracts and DNA binding ELISA assays. In cells of both DLBCL subtypes, silencing of the SMO transcriptional effector GLI1 showed no changes in the activation status of both pathways. The modulation in the activation status of AKT and NF-KB was also detected using SMO inhibitors, cyclopamine-KAAD and HhAntag (Genentech Inc) or activators, purmorphamine and recombinant HH protein. Combinatorial treatments with increasing concentrations of SMO inhibitors (cyclopamine and HhAntag [1.6, 3.2 and 4.8 μM]) with minimal lethal doses of Ly294002 (PI3K inhibitor) or BAY-11 (NF-KB inhibitor) were also performed. Using cell viability, and apoptosis (Annexin-V) assays, we found that combined treatments of PI3K or NF-KB inhibitors with a SMO inhibitor resulted in an additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatments with SMO inhibitors alone. Taken together, our data shed novel light on the contribution of SMO on the activation of PI3K/AKT and NF-kB pathways in DLBCL. Our data also provide a rationale to use SMO inhibitors in combination with inhibitors of other oncogenic pathways such as PI3K/AKT and/or NF-KB for the treatment of patients with DLBCL. Disclosures:No relevant conflicts of interest to declare.