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: Alterations in cancer cell metabolism is a century old concept recently recognized as one of the hallmarks of cancer. Cancer cells, including multiple myeloma (MM), largely shift how they utilize the glucose they consume to glycolysis from that of oxidative phosphorylation (OXPHOS). This phenomenon supports the cancer cell’s large anabolic demands for continuous growth and proliferation and is reinforced by key signaling pathways. However, as a cancer of the previously non-dividing plasma cell, these metabolic adaptations are only beginning to be documented in MM. CD28 is classically known as the T-cell co-stimulatory receptor, but is also expressed on normal plasma cells and their malignant counterparts. Previous data suggests that CD28 is required for MM cell survival, protective during stress induced conditions, and correlates with poor prognosis in the clinic. Furthermore, studies using the anti-CD28 activating monoclonal antibody (mAb) have identified phosphatidyl-inositol 3-kinase (PI3K)/Akt activation to be a key driver of its pro-survival function. Akt is also an important integrator of cellular metabolism and cell growth and proliferation signaling pathways. Therefore, its CD28 mediated activation may uncover the mechanism by which MM cells are able to metabolically sustain stress induced conditions and thrive thereafter. Herein, we show that when CD28 is activated by anti-CD28 mAb (10µg/ml) under stressful conditions (media serum reduction), Akt (T308) phosphorylation increases, resulting in an increase of total protein and cell surface expression of the glucose transporter, GLUT1. To assess if cells take in more glucose in the presence of anti-CD28 mAb they were cultured in glucose free media and glucose was then added back at concentrations of 0.5, 1 and 5mM with or without activating anti-CD28 mAb. Significant increases in glucose uptake were seen in the 5mM anti-CD28 mAb treatment group when compared to the untreated control, correlating positively with the increase in GLUT1 protein expression. To evaluate whether or not CD28 activation induces a preferential for glycolytic breakdown of glucose, the same treatment conditions were repeated and lactate production/oxygen consumption, as a measure of glycolysis and OXPHOS respectively, were measured in a fluorometric kinetic assay. Lactate production significantly increased in MM cells treated with anti-CD28 mAb compared to untreated controls, confirming its role in enhancing glycolysis for cell growth and survival. This data is further supported by increased cell death observed in murine MM cells treated with the glycolysis inhibitor, 2-Deoxyglucose (2-DG). Interestingly, the level of oxygen consumption was comparable in all groups suggesting not only minimal effect in response to CD28 activation, but also relatively unimpaired OXPHOS in MM cells. Furthermore, analysis of mitochondrial biogenesis using the mitotracker green stain and production of reactive oxygen species by 2’,7’-difchlorofluorescin diacetate (DCFDA) oxidation in murine MM cells also revealed both processes to be intact and increased in the presence of CD28 activation. Taken together, these results suggest that CD28 signaling plays a strategic role in shifting the metabolic axis to that of increased glucose uptake and consumption via glycolysis through phosphorylation of PI3K/Akt and upregulation of GLUT1 expression. Pharmacological inhibition of CD28 is therefore an attractive avenue for therapeutic intervention in MM and we have previously shown that interfering with CD28 and its interacting ligands, CD80/CD86, using the CTLA4-Ig fusion protein, is effective in this regard. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma is an incurable hematological malignancy of transformed plasma cells. Many cellular interactions and soluble factors have been demonstrated to play a role in myeloma pathogenesis; however, novel targets to enhance therapeutic intervention are needed. We have demonstrated that CD28 signaling in myeloma cells supports their survival during chemotherapeutic challenge in vitro and in vivo. However, the cellular mechanisms by which CD28 confers this survival advantage to myeloma cells are not completely understood. CD28 is best characterized as the canonical T cell co-stimulatory molecule. During T cell activation, CD28 signaling induces glycolysis, a metabolic program required for T cell proliferation and functional maturation. In the absence of glycolysis, T cells utilize fatty acid oxidation for energy production through the mitochondria. However, the way in which CD28 regulates metabolism in multiple myeloma is not well understood. Here we present evidence that CD28 signaling induces glut1 expression, and that poisoning the glycolytic pathway inhibits proliferation and survival of myeloma cells. AMPK, an energy sensitive kinase known to regulate metabolism by driving fatty acid oxidation, is normally activated when cellular energy levels are low. Interestingly, poisoning glycolysis with a glucose analogue that cannot be processed (2DG) leads to AMPK inhibition in myeloma cells. Furthermore, pharmacological activation of AMPK by AICAR, an AMP analogue, is not sufficient to rescue myeloma cell proliferation from glycolytic inhibition and in fact increases cell death (p