CD28 Induces Mitochondrial Respiration through Slp-76 in Long-Lived Plasma Cells for Reactive Oxygen Species-Dependent Survival
Authors: Utley Adam T. , Ventro Daniela , Murray Megan , Mahpour Amin , Lee Kelvin P. 
Published: October 2019

Abstract
Sustained humoral immunity is dependent upon the production of neutralizing antigen-specific antibodies by plasma cells. Upon antigen activation, B cells differentiate into short-lived plasma cells (SLPCs) which home to secondary lymphoid organs such as the spleen where they live for days to weeks before dying by apoptosis. However, there are examples in the human population in which neutralizing antibody titers persist despite a lack of continual antigen exposure. Therefore an expanded model has been proposed wherein activated B cells differentiate into long-lived plasma cells (LLPCs) that home to the bone marrow and survive indefinitely in specialized survival niches. They are not intrinsically long-lived, but rather depend upon extrinsic survival signals for their persistence and long-term antibody production. Furthermore, many of these survival signals are shared with their malignant counterpart in Multiple Myeloma (MM). Our work centers on the cellular and molecular mechanisms by which MM and LLPCs survive in the bone marrow microenvironment.
Our lab has recently demonstrated that CD28 provides an intrinsic survival signal to LLPCs. Plasma cells deficient in CD28 or mutated in the CD28-Grb2/Vav binding region don't survive and cannot sustain antigen-specific antibody titers post-vaccination. CD28 also provides a survival signal in MM under chemotherapeutic stress. CD28 is best characterized as the canonical co-stimulatory molecule in T cells governing metabolic fitness. Plasma cells and MM are highly biosynthetic and require metabolic expenditure. However, the molecular and metabolic pathways that provide this CD28-mediated survival advantage in LLPCs/MM are not well understood.
Here we demonstrate that CD28 enhances mitochondrial membrane potential in LLPCs and MM, but not in SLPCs. In addition, CD28 induces mitochondrial biogenesis in LLPC and MM. By using oxygen consumption as a direct readout of mitochondrial respiration, we show that CD28 activation increases oxidative phosphorylation. A major byproduct of respiration is the production of reactive oxygen species (ROS). CD28 activation induces mitochondrial-derived ROS in LLPCs but not SLPCs, as seen using the mitoROS specific dye MitoSox. Furthermore, in the mutant LLPCs that cannot signal through CD28-Grb2/Vav, CD28 cannot induce ROS.
ROS are well-characterized cell-damaging agents. In order to elucidate the effects of ROS on LLPCs, we specifically inhibited ROS produced by the mitochondria with MnTBAP. Paradoxically, inhibiting ROS prevents CD28-mediated survival. Taken together the data suggest that CD28 increases mitochondrial respiration for ROS-dependent survival. By analyzing open-source RNAseq data acquired from purified populations of bone marrow LLPCs and splenic SLPCs, the LLPC population exhibits higher transcript levels of genes associated with mitochondrial respiration, oxidative phosphorylation, and the TCA cycle. However, the signals that drive this metabolic program are not characterized.
Building upon our previous findings that the CD28 Grb2/Vav binding moiety on the cytoplasmic tail is required for CD28-mediated survival, we interrogated this signaling axis. Upon CD28 activation, Vav colocalizes to the CD28 receptor seen via ImageStream technology. A major adaptor protein downstream of Vav, Slp-76, is highly expressed in LLPCs and MM but not in SLPCs at the protein level as evidenced by intracellular flow cytometry. By using a transcriptomic approach, Slp-76 is also highly upregulated in human LLPCs compared to SLPCs. We then examined the ability of CD28 to provide a prosurvival signal via genetic approaches wherein Slp-76 was knocked-out or mutated at the Vav binding region. In either case, CD28 was not capable of inducing survival to LLPCs. When examining the knock-in mice that cannot signal through Vav/Slp-76, the LLPC population with high levels of ROS is completely absent when compared to wild type LLPCs. Interestingly, CD28 upregulates Slp-76 at mRNA and protein level in both SLPC and LLPC, suggesting that CD28 may be capable of determining plasma cell fate at the onset of differentiation.
Taken together the data suggests that CD28 may regulate LLPC induction, survival and function through Grb2/Vav/Slp-76 mediated metabolic reprogramming. This makes CD28 an attractive target in vaccine design and for treatment of both autoimmune disorders and multiple myeloma.

Disclosures
No relevant conflicts of interest to declare.