CD28 Regulates Mitochondrial Metabolism in Long Lived Plasma Cells for Survival
Authors: Utley Adam T , Rozanski Cheryl H , Carlson Louise M , Lee Kelvin P 
Published: October 2019

Abstract
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.