Browsing by Subject "B cell"

Burkitt lymphoma (BL) is an aggressive germinal center (GC) B cell derived lymphoma. BL accounts for 40% of pediatric lymphoma cases in the United States and over half of all pediatric malignancies in Sub Saharan Africa. BL is characterized by the t(8;14) chromosomal translocation that results in MYC overexpression. Translocation of MYC alone is insufficient to induce lymphomagenesis; additional genetic mutations are required. A better functional characterization of the genetic drivers of BL will lend insight into pathways that drive Burkitt lymphomagenesis, providing an opportunity to identify novel drug targets and develop improved therapies.

In order to identify novel genetic drivers of BL our lab previously sequenced 101 BL tumors with paired normal samples. We found that DDX3X is recurrently mutated in 46% of BL tumors, making it the third most commonly mutated gene in BL. We observed that DDX3X mutations are either truncating mutations (22%) or missense mutations (88%) that cluster around the two highly conserved functional domains of the protein. Based on the non-focal distribution of missense mutations throughout the DDX3X coding sequence and the high presence of truncation mutations in BL tumors, we hypothesize that in GC B cells DDX3X acts as a tumor suppressor gene whose normal function is destroyed by BL associated mutations, facilitating lymphomagenesis.

While DDX3X is frequently mutated in many types of cancer, its role in malignancy is poorly understood. In this study we focused on elucidation of the role of DDX3X in the specific context of GC B cells from which BLs arise. To test the hypothesis that DDX3X normally functions as a tumor suppressor we modeled DDX3X deficiency in normal and malignant GC B cells using parallel in vitro and in vivo approaches. First, we created transgenic Ddx3x deficiency mouse models with and without MYC overexpression. This approach allowed us to study DDX3X in a system that models the complexities of the immune system on a genetically defined background. Second, we used genome editing to precisely delete DDX3X expression in BL cell lines. BL is a genetically complex disease and the the use of BL cell lines provides allowed us to study the role of DDX3X on a genetic background typical of BL tumors. We then characterized both DDX3X loss of function models with respect to cellular processes related to tumor development. Third, we performed cross-linking immunoprecipitation with sequencing (CLIP-Seq) to identify the RNA targets of DDX3X in the germinal center.

Our study lead to unexpected results regarding the contribution of DDX3X to Burkitt lymphomagenesis, and highlighted an important role for DDX3X in B cell development. We found that Ddx3x deficiency in a mouse model of BL increased the time to tumor development by reducing the global B cell population available for malignant transformation. In tandem we found that Ddx3x deficiency at the GC B cell stage significantly reduced the GC B cell population in multiple lymphoid tissues, regardless of MYC status. Interestingly, we found that Ddx3x deficiency in pre-B cells expanded the pre-B cell population but decreased the population of later B cell stages. We then confirmed that the reduction of GC B cells in response to Ddx3x deficiency was not due to defects in GC B cell migration, germinal center architecture, cell cycle progression, or apoptosis. These combined data suggest an essential function for DDX3X in B cell development, particularly at the pre-B cell and GC B cell stages.

Similar to in the mice, in cell culture we also found that DDX3X loss did not significantly alter apoptosis or cell cycle progression. We found some evidence that DDX3X may play a role in DNA damage repair but these results were not consistent across conditions. Lastly, we identified DDX3X targeted RNA binding partners using CLIP-Seq. Our data corroborates previously published CLIP-Seq experiments showing that DDX3X binds numerous RNAs involved in translation and RNA processing. Additionally, we identified for the first time that DDX3X binds RNAs falling in the BRCA1 and ATM gene sets. Further experimentation is needed to determine the role DDX3X plays in these pathways with relationship to Burkitt lymphomagenesis.

The interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes.

Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.

Understanding B-lymphocyte fate specification during B-cell development and humoral responses is important for developing vaccines and tools for studying B cells.

In the first study, I elucidated major factors influencing the fate of 2F5 B-cell receptor (BCR) knockin B cells as they traverse immune tolerance checkpoints. 2F5 is an HIV-1 broadly neutralizing antibody (bnAb) whose neutralizing activity is linked to its ability to bind two autoantigens: host-derived viral membrane phospholipids and the ELDKWA epitope found in both the viral envelope (Env) protein and in the host enzyme, kynureninase (KYNU). The development of 2F5-like B cells is proscribed by immune tolerance controls, but it is unknown whether tolerizing counterselection is driven primarily by lipid- or by KYNU-specificity (or by both equally). It is also unclear whether BCR editing purges the 2F5 BCR from the peripheral repertoire of 2F5 knockin mice. Answering these questions is important for evaluating potential HIV-1 vaccine strategies.

I sampled thousands of B cells from before and after the first and second tolerance checkpoints in 2F5 BCR knockin mice. After culturing individual B cells in an in vitro system that supports B-cell proliferation and differentiation into Ab-secreting cells, I determined the specificities of the secreted clonal IgGs, and recovered the V(D)J rearrangements encoding these Abs. I found that in 2F5 knockin mice, nearly all (pre-tolerance) small pre-B cells express the knockin heavy chain and light chain (LC), and avidly bind Env, KYNU, and a model lipid, cardiolipin. In contrast, extensive LC editing completely purges the (post-tolerance) mature B cell compartment of Env- and KYNU-reactivity, although cardiolipin-reactivity remains mostly intact. Thus, I conclude that tolerization of 2F5 B cells is driven primarily by KYNU-reactivity, and occurs in large part due to LC editing. Remarkably, peripheral anergic B cells (B220+IgM-IgDhi) are enriched for Env- and KYNU-reactivity, and express a restricted repertoire that partially overlaps with that of mature B cells, indicating that peripheral fate determination is at least partly stochastic. The data support that activation of anergic B cells may be a viable route for HIV-1 vaccination.

In the second study, I tested whether transcriptional re-programming can force activated mature B cells to sustain B-cell identity and block terminal differentiation into plasma cells (PCs). Controlling B-cell terminal fate commitment has numerous potential applications in science and medicine, but the means to do so have remained elusive. PC differentiation is governed by a transcription factor network comprising Pax5, Bcl6, and Bach2 – which promote B-cell identity and antagonize PC differentiation – and IRF4 and Blimp1, which cooperate to extinguish B-cell identity and coordinate PC differentiation.

I generated mouse primary B cells harboring gain-of-function in Pax5, Bach2, or Bcl6, or loss-of-function in IRF4 or Blimp1, and then continuously stimulated these B cells with CD154 and IL-21 in vitro. I found that transgenic expression of Bach2 or Bcl6 prohibits PC commitment, maintains markers of B-cell identity, and endows B cells with extraordinary growth potential in response to T-cell help signals. Long-term Bach2-transgenic B cell lines have genetically stable BCRs, express high levels of MHCII and molecules for co-stimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established cell line causes the cells to secrete large quantities of immunoglobulin. These results provide insight into molecular control over activated B-cell fate, and suggest that enforced expression of Bach2 in vivo may augment germinal center (GC) B cell or memory B cell (MBC) differentiation at the expense of PC commitment. Additionally, culturing Bach2-transgenic B cells has potential applications in monoclonal Ab production, BCR signaling studies, T-cell epitope-mapping studies, and more.

Next, I generated a new knockin mouse harboring a doxycycline-inducible Bach2 transgene and a fluorescent reporter protein, and I used this mouse strain to determine how enforced expression of Bach2 influences the fate of B cells activated in vivo. Constitutive Bach2 expression had no impact on the total number of GC B cells or MBCs in the draining lymph node at 12 days post-immunization, but reduced local PC numbers by ~10-fold. Interestingly, B cells expressing transgenic Bach2 were ~2-fold enriched among GC B cells and MBCs relative to their frequency among mature follicular B cells. Additionally, populations of GC B and MBCs expressing transgenic Bach2 contained significantly higher frequencies of cells brightly stained by fluorescent antigen. As these antigen-bright cells are presumed to have the highest-affinity BCRs, which would normally pre-dispose them toward PC differentiation, I tentatively conclude that enforced expression of Bach2 prevents GC B cells from differentiating into PCs, and instead causes them to remain in GCs or join the MBC pool.

The inducible Bach2-knockin mouse also made it practical to attempt high-throughput isolation of long-lived, monoclonal, antigen-specific B cell lines. By isolating and culturing individual follicular and GC B cells constitutively expressing transgenic Bach2, I generated >20 long-lived cell lines, of which two expressed immunogen-specific BCRs. On withdrawing doxycycline and silencing the Bach2 transgene, all of the cell lines rapidly differentiated into PCs and secreted large quantities of Ig. Thus, inducible Bach2 mice may be an attractive tool for cloning antigen-specific B-cell lines.

Several HIV-1 neutralizing antibodies (e.g. 2F5, 4E10) have been shown to react with self-antigens, suggesting that effective humoral responses to HIV-1 may be constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. I have tracked the development of 2F5-like HIV-1 gp41 membrane proximal external region (MPER)-reactive B cells throughout ontogeny using B-cell tetramer reagents. In BL/6 mice, MPER-binding populations are lost during normal B-cell development and immunization with HIV-1 MPER antigen does not elicit robust humoral responses. I have identified Kynureninase as a self-antigen that is recognized by 2F5 antibody and, therefore, is a molecule that could mediate the developmental loss of B cells reactive to an epitope shared by HIV gp41 and Kynureninase. To recover these MPER-reactive cells, I describe and characterize a stromal-cell independent culture system that efficiently supports pro-B cell to IgM+ B-cell development with near normal levels of IgH and Igkappa diversity. B-cell development in vitro closely follows the patterns of development in vivo with culture derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. Immature and transitional B-cell compartments are reduced, due to the induction of tolerance, in the bone marrow of 3H9 IgH knockin mice ; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4-/- animals following the reconstitution of RAG-1-/- mice with IgM+ CD cells derived from BL/6 mice. I show that HIV-1 MPER-reactive B cells are recovered from both BL/6 and 2F5 IgH knockin bone marrow using this in vitro culture system. RAG-1-/- mice reconstituted with these culture-derived B and T cells generate strong germinal center and antibody responses to HIV-1 MPER antigens. These data demonstrate that the humoral immune response to this HIV-1 gp41 MPER antigen can be restored in mice when the constraints of B-cell tolerance have been relaxed.

Epstein-Barr Virus (EBV) is a ubiquitous gamma-herpesvirus in the human population and highly associated with lymphomas of the immune-suppressed. EBV maintains itself within the human host via temporal regulation of viral gene expression before establishing latency in resting memory B cells. Experimentally, we can model these immune-compromised lymphomas via in vitro infection of B cells isolated from peripheral blood. This infection model is biphasic where the initial phase is characterized by hyper-proliferation and expression of the EBV nuclear antigens (EBNAs) in the absence of the latent membrane proteins (LMPs) called latency IIb before transitioning to the NFkB-dependent latency III lymphoblastoid cell line (LCL) stage characterized by full expression of the LMPs in the presence of the EBNAs. This temporal regulation manipulates host cell proliferation rates and metabolic profiles of infected B lymphocytes. Our laboratory and my studies, in particular, are focused on the latency IIb to latency III transition as these states are often observed in patient biopsies of EBV-associated malignancies.

Latent membrane protein 1 (LMP1) expression defines latency III and has been used in lymphoma samples to identify EBV latency III in vivo. While LMP1 expression is lower in latency IIb, the mechanism by which it is repressed is currently unknown. Additionally, while LMP1 expression can be used to distinguish latency IIb from latency III, LMP1 expression varies widely within latency III and a subset of LMP1lo latency III cells express LMP1 at levels similar to latency IIb making these cells hard to distinguish in vitro and in vivo. Lastly, while EBV manipulates host cell metabolism, the viral and host factors important for metabolic rewiring have not been fully elucidated.

Using RT-qPCR, ChIP-qPCR, RNA-sequencing, Western Blotting, flow cytometry, and bioinformatic approaches we have identified c-Myc as a repressor of LMP1 during latency IIb. We have also identified host mRNA biomarkers capable of distinguishing latency IIb from latency III. This discovery was additionally leveraged to develop a multiplex RNA-FISH platform capable of distinguishing latency IIb from latency III. Lastly, we have identified host and viral proteins necessary to rewire host cell metabolism and sustain EBV-mediated B cell proliferation. This work, taken together, adds new understanding to EBV latency establishment, heterogeneity, and host B cell biology to develop new, more targeted, therapeutics for EBV-associated lymphomas in the future.