ALBERT

Description: Sustained humoral immunity is dependent upon the ability of plasma cells to produce antigen specific antibody titers over a long period of time. Whether fighting against pathogens such as Ebola, Influenza, or the common cold, the continual presence of neutralizing antibodies in circulation is critical for an effective humoral immune response. When activated by antigen, B cells differentiate into a short lived plasma cell (SLPC) pool and reside in secondary lymphoid organs such as the spleen for days to weeks before dying by apoptosis. However, in the absence of constant antigenic presence there are examples of continual production of antigen-specific antibodies in the human body. To explain this, a different subset of long lived plasma cells (LLPC) has been proposed wherein the plasma cells compete for space in specialized bone marrow niches. These cells are not intrinsically long lived, but rather depend upon extrinsic survival factors for their persistence. Many of the survival factors for LLPCs in the bone marrow are shared with their malignant counterpart, e.g. multiple myeloma (MM). Our work centers on the elucidation of mechanisms by which both MM and LLPCs survive in the bone marrow microenvironment. Recently our lab has demonstrated a cell intrinsic role for CD28 signaling in the survival of both LLPCs and MM. CD28 is known as the canonical T cell co-stimulatory molecule and is required for effector T cell metabolic fitness. Under nutrient deprivation and chemotherapeutic challenge, CD28 is able to induce survival of LLPCs and MM cells respectively. However, the molecular and metabolic pathways that govern this prosurvival effect are not well understood. Here we demonstrate that CD28 induces mitochondrial respiration in bone marrow resident LLPCs but not in splenic SLPCs by staining with a mitochondrial-specific dye that is taken up in proportion to the mitochondrial membrane potential. A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). As a result of increased mitochondrial metabolism through the electron transport chain, CD28 is able to induce ROS in LLPCs but not in SLPCs. This is somewhat counterintuitive, in that ROS are well-characterized as cell damaging agents. Fascinatingly, mitochondrial respiration dependent ROS production downstream of CD28 is required for the prosurvival effect seen in LLPCs. Mechanistically, CD28-mediated production of ROS drive NFkB translocation as seen by ImageStream technology, which goes on to drive Blimp1 expression, the master transcriptional regulator of plasma cell identity. Utilizing a luciferase expressing plasmid and including different lengths of the Blimp1 promoter, we show that the CD28 responsive element lies from 4500 to 7500 base pairs from the transcriptional start site. Furthermore, we are able to demonstrate by CHIP that NFkB binds directly to the Blimp1 promoter. In order to understand why this occurs in MM and bone marrow resident LLPCs but not splenic derived SLPCs, we made use of in silico and genetic approaches to discover how the cells differentially signal through CD28. We demonstrate that the Grb2-Vav binding domain in the cytoplasmic tail of CD28 is critical for its prosurvival signal. Vav is known to bind the major adaptor molecule Slp-76. Using transcriptomic analysis we demonstrate that in humans, the major adaptor molecule Slp-76 is highly expressed in LLPCs but not SLPCs. A major downstream target of Slp-76 is PLC-g1 which is phosphorylated by CD28 activation in LLPCs but not SLPCs. To demonstrate that signals emanating from Slp-76 drive LLPC survival, we made use of Slp-76 mutant mice wherein the Vav binding domain is mutated. These mice had lower LLPC numbers and could not transduce a CD28 prosurvival signal. Furthermore, genetic knockdown of Slp-76 diminished the ability of CD28 to induce Blimp1 upregulation. Altogether these data suggest that CD28, through a Grb2-Vav-Slp-76 signal induces mitochondrial respiration dependent production of ROS. These ROS go on to activate NFkB mediated induction of Blimp1, thereby reinforcing the plasma cell phenotype for survival and function. This knowldedge will augment our ability to create effective vaccines as well as disrupt antibody-mediated autoimmunity and multiple myeloma progression in patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1737 Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, is has been shown normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment including cell-to-cell interactions. Leading us to rationalize, modulating this interaction could either enhance antibody production for cancer vaccine development or conversely compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC plays an essential role. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases the survival and IgG production of LLPC. These observations led us to directly investigate the role of CD28 in LLPC survival as well as cell-cell interactions with CD80/CD86+ bone marrow derived dendritic cells (BMDC). Utilizing normal murine bone marrow and splenic PC as our model system we further investigated the role of CD28 in LLPC function and survival. We have previously shown, in vitro serum starvation experiments, direct activation of CD28 increased survival of LLPC by 12-fold (p

Description: Abstract 473 Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable for most patients. Myeloma cells are the transformed counterpart of the normal, bone marrow-resident long lived plasma cells (LLPC) that can survive for years to decades and are also responsible for long-term production of protective antibody titers. Critical interactions between MM and their bone marrow stromal cells (BMSC), that are important for their long term survival and chemotherapy resistance (in MM) identifies potential therapeutic targets, and are often the target for many IMIDs (thalidomide, lenalidomide). However, the specific molecular and cellular components of these interactions remain poorly characterized. These interactions directly transduce pro-survival signals to the myeloma cells as well as induce niche production of supportive soluble factors, the prototypic example being MM induction of stromal IL-6 - a key pro-MM survival cytokine. Despite this importance, the specific molecular and cellular components involved in these interactions remain poorly characterized. We have previously shown that CD28 expressed on human myeloma cells directly transduces a survival signal to MM cells upon binding its CD80/CD86 ligands on conventional (myeloid) dendritic cells (DC), and that DC preferentially co-localize with myeloma cells in the patient bone marrow niche. In our previous presentation at ASH (2010), we showed that myeloma cells interact with DCs to produce immunosuppressive factors such as IDO, and protects myeloma cells against cell death via a CD28-B7 mediated interaction. We now show that DC-IDO not only suppresses T-cell proliferation in invitro assays, but also contributes to the immunosuppressive milieu by inducing naïve T-cells to form T-regs (Fig 1). We hypothesize that while IDO activity by itself can suppress T-cell proliferation and induce T-cell growth arrest and apoptosis, the generation of T-regs by these immunosuppressive DCs (previous studies have shown a close association of T-regs with myeloma cells) form two facets of an immunosuppressive defense that myeloma cells mount against the body's anti-myeloma immune response.Figure 1Figure 1. As we have shown in our earlier presentations at ASH, on the myeloma side, the activation of CD28 induces pro-survival responses that can be extinguished by blocking CD28-B7 interactions between myeloma cells and DCs. Now we show that CD28 activation is accompanied by rapid tyrosine phosphorylation of CD28, association of p85 (PI3K), activation of Vav-1 and increase in CD28 associated tyrosine kinase activity, as shown by immunoprecipitation, western and kinase activity assays. Our data suggests a role for SLP76 downstream of Vav1 in CD28 mediated survival of myeloma cells. Immunoassays with protein extracts from myeloma cells that were previously co-cultured with DCs and isolated using positive magnetic selection show a decrease in BLIMP1 expression (Fig 2) that correlates with published data by other groups that indicates that DC mediated increase in myeloma clonogenicity/tumorigenicity is accompanied by increases in BCL6 (a negative regulator of BLIMP1). Blocking CD28-B7 interactions between myeloma and dendritic cells reversed this decrease in BLIMP1 expression. The implications of this to myeloma survival is currently under study in our lab.Figure 2Figure 2. In summary, we propose that CD28 expressed by myeloma cells serves as a central molecular bridge within a complex and integrated cellular and soluble factor microenvironment necessary for MM cell survival. CD28 directly delivers a pro-survival signal to the myeloma cell, and by ligating CD80/CD86 on conventional DC backsignals to these stromal cells to elicit immunosuppressive enzyme IDO and inducing immunosuppressive T-regulatory cells. Although undoubtedly incomplete, this model begins to point to novel therapeutic targets for the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 132 Multiple myeloma (MM), an incurable neoplasia of terminally differentiated plasma cells, are critically dependent on their interactions with bone marrow stromal cells (BMSC) for essential survival signals, growth and immunosuppressive factors. Very little is known about the specific BM cell type or the molecular elements in these interactions, an understanding of which could provide novel targets that could be interdicted to enhance conventional chemotherapy. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma tumoral expansion. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with BM cells that express B7 (CD80/CD86) such as dendritic cells (DCs, that are known to be closely associated with MM cells in the BM) or with CD86+ MM cells themselves. We had previously shown (ASH2008, #I-769) that blocking CD28-CD86 interactions between myeloma cells with high affinity B7 ligand CTLA4Ig (Abatacept®) sensitized myeloma cells to chemotherapy. Now we show that myeloma cells co-cultured with myeloid DCs in vitro derive both direct and indirect survival signals from DCs, and this can be partially blocked by commercially available reagents. Our data show that flow cytometric analysis of mononuclear cells (MNC) from BM aspirates of myeloma patients with increased CD138+ plasma cell populations (9-58%), show an increased CD11b+ (myeloid) population (20-37%) as well, which is in contrast to healthy transplant donor controls (12-15% CD11b+, 4–6% CD138+). Moreover, a larger fraction (11-47%) of the myeloma CD138+ plasma cells expressed CD28 compared to healthy control (3.3-7.7%). Also, when we analyzed gene expression datasets (NCBI #GSE5900 and GSE4204) from plasma cells (PC) of normal donors, monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM) and newly diagnosed multiple myeloma (MM), we found a progressive increase in patients showing CD28 expression with increasing severity of disease (normal

Description: Abstract 182 Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment, including cell-to-cell interactions. This lead us to hypothesize that modulating these interactions could either enhance antibody production for vaccine development or, conversely, compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown that the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC, plays an essential role in survival. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases survival and IgG production in LLPC. Consistent with these findings, we show in vivo, vaccinated bone marrow CD28−/−:μMT chimeras had significantly reduced long-term antibody titers and decreased LLPC (but not SLPC) t1/2 from 426 to 63 days. These findings demonstrate the existence of a distinct bone marrow (BM) LLPC subset necessary to sustain antibody titers, and establish a central role for CD28 function in the maintenance of plasma cells and humoral immunity. While CD28 signaling has been shown to play an important role in maintaining long-term humoral immune responses, the mechanism by which CD28 signaling affects PC function has not yet been determined. To further elucidate CD28 signaling in BM PC, we utilized CD28 conditional knock-in mice. In these mice, the CD28 cytoplasmic tail is mutated at either the YMNM or proline-rich motifs, resulting in an inhibition of PI3K or vav signaling, respectively. We found that CD28-vav signaling deficient BM PC were selectively depleted in vivo and could not be rescued by CD28 activation in in vitro serum starvation conditions. Furthermore, anti-CD28 mAb drove a 1.5 fold increase in Blimp-1 expression in BM PC, compared to control. This increase was regulated through the CD28-vav signaling pathway, as CD28 activation in CD28-vav signaling deficient BM PC did not increase Blimp-1 expression. To further determine if CD28 is acting directly on the Blimp-1 promoter, we examined in silico for a CD28RE composite element, previously reported to transcriptionally regulate IL-2 production in T cells and IL-8 production in myeloma cells. To our surprise, we found a CD28RE “like” site 4712bp upstream of the Blimp-1 start site. To confirm CD28 transcriptionally regulates Blimp-1 promoter activity, we transfected the CD28+ plasmacytoma cell line J558 with full-length or truncated Blimp-1 promoter constructs (i.e. 7000bp, 4500bp, 1500bp). We found CD28 activation enhances Blimp-1 activity in J558 cells transfected with full-length-Blimp-1, and this activity was lost when the promoter was truncated. Using site-directed mutagenesis, we confirmed the CD28RE is required for induction of Blimp-1 in PC. Furthermore, we show CD28 activation of Blimp-1 increases the BCMA receptor in BM PC. Taken together, our data suggests the CD28-vav signaling pathway in PC induces a CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC and humoral immunity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1741 Multiple myeloma is a neoplasm of bone marrow resident plasma cells characterized by critical interactions between myeloma cells and bone marrow stromal cells. This interaction leads to production of IL-6, an important factor in myeloma cell biology. However, the molecular and cellular components involved in myeloma induced IL-6 production remain largely uncharacterized. While at the cellular level, dendritic cells (DC)-expressing CD80/CD86 (collectively called B7, ligands with short cytoplasmic tails and signaling partners of CD28 expressed on myeloma cells) - in the bone marrow microenvironment have been implicated as being an important component, at the molecular level the CD28-B7 and Notch1-Jagged2 pathways were separately implicated by us (in DC) and others in myeloma induced IL-6 production. Although Notch signaling leading to IL-6 production in DC is well understood, the mechanism of “backsignaling” via B7 is largely uncharacterized. To better understand downstream B7 signaling leading to IL-6 production, DC were stimulated with CD28-Ig (a soluble form of CD28 which mimicks myeloma cell-bound CD28) in the presence or absence of an inhibitor of Notch signaling, gamma secretase inhibitor (GSI). DC treated with CD28-Ig alone produced significantly (p〈 0.001) higher levels of IL-6 when compared to DC treated with CD28-Ig and GSI. GSI specifically targeted Notch signaling as observed by decreased expression of Notch gene targets: Hes-1 (2 fold decrease) and Deltex-4 (4 fold decrease). Also, decreased IL-6 levels in presence of GSI were not due to the decrease in B7 expression on DC. To specifically implicate the importance of Notch1 and Jagged2, we blocked Notch1 signaling using blocking antibodies and observed a similar decrease in IL-6 production upon blocking Notch1 signaling. Our results suggest that CD28 mediated IL-6 production is dependent on Notch1 signaling and crosstalk between the Notch1-Jagged2 and CD28-B7 pathways leads to IL-6 production by DC. The model of crosstalk between CD28-B7 and Notch1-Jagged2 pathways was also observed in murine bone marrow derived dendritic cells (BMDC), where a significant (p

Description: The long term generation of protective antibodies (Abs) requires the continuous survival of long-lived plasma cells that are maintained within specialized bone marrow niches by complex interactions that remain largely uncharacterized. Previous studies have shown that the T cell costimulatory receptor CD28 is expressed on normal and transformed (murine plasmacytoma, human multiple myeloma) plasma cells – however, its role in the B cell lineage remained unclear. We have recently shown that CD28 expressed on transformed human plasma cells (multiple myeloma cells) directly delivers pro-survival signals to the myeloma cells and protects them against intrinsically and extrinsically induced death (Bahlis et al, 2007). Furthermore, myeloma cells directly interact with dendritic cells (DC, both in vitro and in patient bone marrow biopsies), and the DC provide the ligands (i.e. CD80 and CD86) for myeloma-CD28. Others studies utilizing competitive bone marrow reconstitution have indirectly suggest a role for CD28 in the function and/or survival of normal murine plasma cells (Delogu et al, 2006). These observations led us to directly investigate the role of CD28 in normal plasma cell survival as well as cell-cell interactions with CD80/CD86+ bone marrow derived dendritic cells (BMDC). In vitro serum starvation experiments, direct activation of CD28 by an agonistic anti-CD28 mAb increased survival of serum-starved PC by 63% (p