Survival Of Long-lived Plasma Cells Research Articles

New insights into the ontogeny, diversity, maturation and survival of long-lived plasma cells

New insights into the ontogeny, diversity, maturation and survival of long-lived plasma cells

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

A PRoliferation-Inducing Ligand (APRIL) in the Pathogenesis of Immunoglobulin A Nephropathy: A Review of the Evidence.

A PRoliferation-Inducing Ligand (APRIL), the thirteenth member of the tumor necrosis factor superfamily, plays a key role in the regulation of activated B cells, the survival of long-lived plasma cells, and immunoglobulin (Ig) isotype class switching. Several lines of evidence have implicated APRIL in the pathogenesis of IgA nephropathy (IgAN). Globally, IgAN is the most common primary glomerulonephritis, and it can progress to end-stage kidney disease; yet, disease-modifying treatments for this condition have historically been lacking. The preliminary data in ongoing clinical trials indicate that APRIL inhibition can reduce proteinuria and slow the rate of kidney disease progression by acting at an upstream level in IgAN pathogenesis. In this review, we examine what is known about the physiologic roles of APRIL and evaluate the experimental and epidemiological evidence describing how these normal biologic processes are thought to be subverted in IgAN. The weight of the preclinical, clinical, and genetic data supporting a key role for APRIL in IgAN has galvanized pharmacologic research, and several anti-APRIL drug candidates have now entered clinical development for IgAN. Herein, we present an overview of the clinical results to date. Finally, we explore where more research and evidence are needed to transform potential therapies into clinical benefits for patients with IgAN.

Non-canonical unfolded protein response transcription factor CREB3L2 regulates survival of long-lived plasma cells

Abstract Differentiation of activated B cells into antibody-secreting cells (ASCs) involves extensive endoplasmic reticulum (ER) & Golgi biogenesis and remodeling that enable increased antibody synthesis and secretion. These processes are mediated by the unfolded protein response (UPR). Canonical UPR pathway genes are regulated by the transcription factors (TFs) XBP-1 and ATFs, which are activated upon ER or Golgi stress. Using scRNA-seq to analyze the generation of splenic and bone marrow (BM) plasma cells (PCs), we discovered that the Creb3l2gene, encoding a non-canonical UPR TF, is specifically expressed in PCs, with highest levels in long-lived PCs (LLPCs). CREB3L2 has been shown to regulate transcription of genes encoding components critical to secretory cells of the pancreas and pituitary gland. To analyze the function of CREB3L2 in ASCs, we pursued a two-pronged strategy (i) using CRISPR/Cas9-RNP editing in the murine CH12 B lymphoma cell line to generate out-of-frame deletions in exon 2 of the Creb3l2gene (Creb3l2−/−) and (ii) generating Creb3l2B-cell conditional KO (cKO) mice carrying a floxed allele of the Creb3l2gene by inserting loxpsites flanking exon 7 with a CD19-cre driver (Creb3l2fl/flCD19 cre/+). RNA-seq of Creb3l2−/−CH12 cells shows CREB3L2 positively regulated genes including Cd93and Cd28& its signaling adapter SLP-76 (Lcp2), that are involved in plasma cell survival in the BM niche. Expression of CD28/SLP-76 and CD93 proteins were strongly diminished in Creb3l2−/−CH12 cells. Consistent with these findings, Creb3l2cKO mice had reduced NP-specific LLPCs (90 d.p.i) after NP-KLH immunization. Thus, CREB3L2 appears to selectively regulate the survival of LLPCs by controlling the expression of CD28/SLP-76 and CD93. Supported by grants from NIAID (U01 AI141990, R01 AI145064).

Indoleamine 2,3-dioxygenase 1 is essential for sustaining durable antibody responses

Humoral immunity is essential for protection against pathogens, emphasized by the prevention of 2-3 million deaths worldwide annually by childhood immunizations. Long-term protective immunity is dependent on the continual production of neutralizing antibodies by the subset of long-lived plasma cells (LLPCs). LLPCs are not intrinsically long-lived, but require interaction with LLPC niche stromal cells for survival. However, it remains unclear which and how these interactions sustain LLPC survival and long-term humoral immunity. We now have found that the immunosuppressive enzyme indoleamine 2,3- dioxygenase 1 (IDO1) is required to sustain antibody responses and LLPC survival. Activation of IDO1 occurs upon the engagement of CD80/CD86 on the niche dendritic cells by CD28 on LLPC. Kynurenine, the product of IDO1 catabolism, activates the aryl hydrocarbon receptor in LLPC, reinforcing CD28 expression and survival signaling. These findings expand the immune function of IDO1 and uncover a novel pathway for sustaining LLPC survival and humoral immunity.

Plasma cell dynamics in the bone marrow niche

SUMMARYUsing intravital imaging, we report that bone marrow (BM) plasma cells (PCs) are motile. BM PCs exhibit a unique migration pattern, characterized by intermittent periods of high motility and longer stretches of confined migration or arrest. BM PCs accumulate into clusters, which have reduced cell motility. APRIL promotes cluster formation and overall PC motility in the BM. Although CXCL12 and its receptor, CXCR4, promote PC motility in the BM, VLA4 activity promotes arrest. However, blocking either pathway promotes PC egress from the BM. Under steady-state conditions, BM PCs recirculate to other bones and spleen. In older mice, overall PC motility and recirculation increase, and this is correlated with increased CXCR4 expression, which depends on PC age or maturation rather than mouse age. Altogether, these results suggest that changes in PC motility and CXCR4 expression are linked with survival of long-lived PCs in the BM.

Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells

SummaryThe persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells.

Targeting Multiple Myeloma through the Biology of Long-Lived Plasma Cells.

Multiple myeloma (MM) is a hematological malignancy of terminally differentiated bone marrow (BM) resident B lymphocytes known as plasma cells (PC). PC that reside in the bone marrow include a distinct population of long-lived plasma cells (LLPC) that have the capacity to live for very long periods of time (decades in the human population). LLPC biology is critical for understanding MM disease induction and progression because MM shares many of the same extrinsic and intrinsic survival programs as LLPC. Extrinsic survival signals required for LLPC survival include soluble factors and cellular partners in the bone marrow microenvironment. Intrinsic programs that enhance cellular fidelity are also required for LLPC survival including increased autophagy, metabolic fitness, the unfolded protein response (UPR), and enhanced responsiveness to endoplasmic reticulum (ER) stress. Targeting LLPC cell survival mechanisms have led to standard of care treatments for MM including proteasome inhibition (Bortezomib), steroids (Dexamethasone), and immunomodulatory drugs (Lenalidomide). MM patients that relapse often do so by circumventing LLPC survival pathways targeted by treatment. Understanding the mechanisms by which LLPC are able to survive can allow us insight into the treatment of MM, which allows for the enhancement of therapeutic strategies in MM both at diagnosis and upon patient relapse.

CD28 Regulates Metabolic Fitness for Long-Lived Plasma Cell Survival

SUMMARYDurable humoral immunity against epidemic infectious disease requires the survival of long-lived plasma cells (LLPCs). LLPC longevity is dependent on metabolic programs distinct from short-lived plasma cells (SLPCs); however, the mechanistic basis for this difference is unclear. We have previously shown that CD28, the prototypic T cell costimulatory receptor, is expressed on both LLPCs and SLPCs but is essential only for LLPC survival. Here we show that CD28 transduces pro-survival signaling specifically in LLPCs through differential SLP76 expression. CD28 signaling in LLPCs increased glucose uptake, mitochondrial mass/respiration, and reactive oxygen species (ROS) production. Unexpectedly, CD28-mediated regulation of mitochondrial respiration, NF-κB activation, and survival was ROS dependent. IRF4, a target of NF-κB, was upregulated by CD28 activation in LLPCs and decreased IRF4 levels correlated with decreased glucose uptake, mitochondrial mass, ROS, and CD28-mediated survival. Altogether, these data demonstrate that CD28 signaling induces a ROS-dependent metabolic program required for LLPC survival.

CD28 Induces Autophagy in Plasma Cells to Enhance Mitochondrial Respiration and Survival

Abstract Durable humoral immunity relies on the persistence of long-lived plasma cells (LLPC) that continuously produce protective antibodies. The prototypical T cell costimulatory molecule CD28, that is also expressed on LLPC, is critical to their survival but the mechanisms involved remain unclear. We found CD28 activation enhances autophagy in LLPC/multiple myeloma as evidenced by increased level of autophagy marker LC3II, elevated autophagosome numbers and decreased level of autophagic cargo receptor P62. Inhibition of autophagy by 3MA or BafA abolished CD28’s pro-survival effects. Mechanistically, CD28 activation increased the protein level of ATG5, a critical autophagy regulator, even with translation blockade, but this was abrogated by proteasome inhibition. Knocking down atg5 eliminated CD28’s pro-survival effects, suggesting CD28 signaling regulates ATG5 degradation to sustain pro-survival autophagy. CD28 activation significantly increased mitochondrial respiration and blocking autophagy prevented this. Free fatty acids (FFA) can be degraded from cellular lipid droplets (LD) by lipophage, a selective form of autophagy, to fuel mitochondrial respiration. We found CD28 activation decreased LD staining and blocking autophagy prevented this. Blocking lipophagic lipase by lalistat eliminated CD28-induced oxidative phosphorylation and pro-survival effects. Addition of FFA (oleic and palmitic acids) boosted oxidative phosphorylation and restored survival with autophagy inhibition but not with fatty acid oxidation blockade. These data suggest CD28 signaling regulates ATG5 protein degradation to enhance autophagy and this in return produces FFA to sustain mitochondrial respiration and support LLPC survival.

The BCMA:Tomato Mouse – a new reporter to characterize long-lived plasma cells

Abstract Plasma cells provide humoral protection by secreting large amounts of antibodies. The heterogeneity of the plasma cell compartment has recently been demonstrated by functional studies and subpopulation markers, but the distinct properties of long-lived plasma cells (LLPC) are still unresolved. B Cell Maturation Antigen (BCMA) has been described as an essential receptor for LLPC survival. To elucidate the mechanisms of BCMA-mediated survival we established a reporter mouse line that expresses a tdTomato fluorescent protein together with BCMA under the control of the promoter of Tnfrsf17, the gene that encodes BCMA. In this reporter model, we could demonstrate the restricted expression of Tomato and BCMA in plasma cells making this an ideal system for the identification and tracking of plasma cells. Importantly, we observed an increase in the abundance of BCMA:Tomato along with a gradual loss of CD19 expression, delineating a developmental progression from plasmablasts to mature plasma cell subsets. In contrast, plasmablasts generated in vitro display only minimal BCMA induction, confirming the independence of BCMA expression from the Blimp1 master regulator of PC differentiation. In summary, we have developed a new plasma cell-specific reporter model that, together with an inducible BCMA-KO mouse, will allow us to investigate the BCMA-dependent signals of the generation and maintenance of LLPC. This work was supported, in part, by the Deutsche Forschungsgemeinschaft (DFG) through research grants TRR130 to H.-M.J. and W.S. and GRK1660 to H.-M.J.

Factors Affecting Early Antibody Secreting Cell Maturation Into Long-Lived Plasma Cells.

Antibody secreting cells (ASCs) are terminally differentiated cells of the humoral immune response and must adapt morphologically, transcriptionally, and metabolically to maintain high-rates of antibody (Ab) secretion. ASCs differentiate from activated B cells in lymph nodes and transiently circulate in the blood. Most of the circulating ASCs undergo apoptosis, but a small fraction of early ASCs migrate to the bone marrow (BM) and eventually mature into long-lived plasma cells (LLPCs). LLPC survival is controlled both intrinsically and extrinsically. Their differentiation and maintenance programs are governed by many intrinsic mechanisms involving anti-apoptosis, autophagy, and metabolism. The extrinsic factors involved in LLPC generation include BM stromal cells, cytokines, and chemokines, such as APRIL, IL-6, and CXCL12. In humans, the BM CD19−CD38hiCD138+ ASC subset is the main repository of LLPCs, and our recent development of an in vitro BM mimic provides essential tools to study environmental cues that support LLPC survival and the critical molecular mechanisms of maturation from early minted blood ASCs to LLPCs. In this review, we summarize the evidence of LLPC generation and maintenance and provide novel paradigms of LLPC maturation.

Survival of Long-Lived Plasma Cells (LLPC): Piecing Together the Puzzle.

Durable humoral immunity is dependent upon the generation of antigen-specific antibody titers, produced by non-proliferating bone marrow resident long-lived plasma cells (LLPC). Longevity is the hallmark of LLPC, but why and how they survive and function for years after antigen exposure is only beginning to be understood. LLPC are not intrinsically long-lived; they require continuous signals from the LLPC niche to survive. Signals unique to LLPC survival (vs. PC survival in general) most notably include those that upregulate the anti-apoptotic factor Mcl-1 and activation of the CD28 receptor expressed on LLPC. Other potential factors include expression of BCMA, upregulation of the transcription factor ZBTB20, and upregulation of the enzyme ENPP1. Metabolic fitness is another key component of LLPC longevity, facilitating the diversion of glucose to generate pyruvate during times of stress to facilitate long term survival. A third major component of LLPC survival is the microenvironment/LLPC niche itself. Cellular partners such as stromal cells, dendritic cells, and T regulatory cells establish a niche for LLPC and drive survival signaling by expressing ligands such as CD80/CD86 for CD28 and producing soluble and stromal factors that contribute to LLPC longevity. These findings have led to the current paradigm wherein both intrinsic and extrinsic mechanisms are required for the survival of LLPC. Here we outline this diverse network of signals and highlight the mechanisms thought to regulate and promote the survival of LLPC. Understanding this network of signals has direct implications in increasing our basic understanding of plasma cell biology, but also in vaccine and therapeutic drug development to address the pathologies that can arise from this subset.

The surprising role of Indoleamine 2,3-dioxygenase (IDO) in supporting the survival of bone marrow resident long lived plasma cells (LLPC)

Abstract Long lived plasma cells (LLPC) are a key component of protective humoral immunity. The ability of these cells to maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow (BM) microenvironment in which these cells reside. Our lab has demonstrated that the co-stimulatory receptor, CD28, is essential for the survival and function of LLPC. Furthermore, when CD28 on LLPC ligates with CD80/CD86 expressed on DC, it induces upregulation of the enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan (Trp) into kynurenine (kyn), a well-known ligand of the aryl hydrocarbon receptor (AhR). AhR is a nuclear transcription factor that can modulate immune responses and is highly expressed in plasma cells (PC). Our preliminary data reveals that IDO knock-out (KO) mice in comparison to wild type (Wt) mice have a decrease in bone marrow resident PCs and a decrease in overall Ig levels. Additionally, 15 weeks post vaccination IDO KO mice have significantly less antigen-specific long lived antibody titers compared to Wt. It was also observed that interaction with DC both in vitro and in vivo is needed for LLPC survival and function. Moreover, AhR activation by kyn causes an increase in CD28 mRNA in LLPC. This leads us to propose a model where CD28 interaction with CD80/CD86 induces IDO production, which through its metabolite kyn activates AhR which can then regulate CD28 expression- thus creating a mechanism that promotes LLPC survival and function. These findings challenge the traditional idea of IDO as an inhibitory molecule and provide the rationale for investigating its essential role in maintaining LLPC survival and sustained antibody responses.

Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion

Human antibody-secreting cells (ASC) in peripheral blood are found after vaccination or infection but rapidly apoptose unless they migrate to the bone marrow (BM). Yet, elements of the BM microenvironment required to sustain long-lived plasma cells (LLPC) remain elusive. Here, we identify BM factors that maintain human ASC > 50 days in vitro. The critical components of the cell-free in vitro BM mimic consist of products from primary BM mesenchymal stromal cells (MSC), a proliferation-inducing ligand (APRIL), and hypoxic conditions. Comparative analysis of protein–protein interactions between BM-MSC proteomics with differential RNA transcriptomics of blood ASC and BM LLPC identify two major survival factors, fibronectin and YWHAZ. The MSC secretome proteins and hypoxic conditions play a role in LLPC survival utilizing mechanisms that downregulate mTORC1 signaling and upregulate hypoxia signatures. In summary, we identify elements of the BM survival niche critical for maturation of blood ASC to BM LLPC.

CD28 induces survival in long-lived plasma cells through NFκB mediated Irf4 upregulation and mitochondrial respiration-dependent reactive oxygen species production.

Abstract Durable humoral immunity is dependent upon sustained antibody production by long-lived plasma cells (LLPCs). We have published that CD28 is required for LLPC survival and antibody production in vivo; however, the mechanistic basis for this is unclear. In T cells, CD28 is known to induce glycolysis at the expense of respiration. Surprisingly, CD28 induces mitochondrial respiration and biogenesis in LLPCs while also increasing glucose uptake. Paradoxically, respiration-dependent reactive oxygen species (ROS) are required for CD28-mediated survival. Using knock-in mice with mutations in the CD28 cytoplasmic tail, we demonstrate that CD28-Grb2-Vav-Slp76 signaling is required for the survival of LLPCs with high mitochondrial mass and ROS in vivo. Mechanistically, CD28 induces Irf4 in an NFκB-dependent manner. CD28-ROS drive cRel translocation, an NFκB subunit, which can bind directly to the Irf4 promoter. We also describe a super enhancer element further upstream of Irf4 driven by the NFκB subunit p65, induced by CD28 specifically in LLPCs. Furthermore, inhibition of NFκB abrogates CD28-mediated glucose uptake, mitochondrial respiration and biogenesis. Irf4 heterozygous LLPCs have lower mitochondrial mass in vivo, and knock-down of Irf4 decreases the mitochondrial mass. This leads to a model wherein CD28, through Grb2-Vav-Slp-76, reinforces LLPC survival through the induction of mitochondrial biogenesis and respiration. Respiration-dependent ROS then augment NFκB-mediated Irf4 expression through direct promoter activity and a previously undescribed super enhancer element. CD28, therefore, makes an attractive target for the augmentation of vaccine design and in the alleviation of antibody-mediated autoimmunity.

Decreased Expression of B cell Maturation Antigen in Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous group of diseases characterized by hypogammaglobulinemia, leading to recurrent infections. Defect in maturation of B cells into functional plasma cells is the most principal defect in CVID. B cell maturation antigen (BCMA) is an essential molecule for survival of long-lived plasma cells. The aim of this study is to evaluate BCMA expression on B cells as well as count peripheral blood plasmablast cells in CVID patients compared with healthy individuals. Blood samples were collected from 15 CVID patients and 15 age- and sex-matched normal controls. Genomic RNA was extracted from peripheral blood mononuclear cell samples using an RNX-Plus Kit. cDNA was synthesized from extracted RNA using a RevertAid First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template for reverse transcription polymerase chain reaction (RT-PCR) to measure BCMA gene expression by Maxima SYBR Green/ROX qPCR Master Mix Kit (Thermo Scientific). Flow cytometry was use...

Neutrophils releasing IL-17A into NETs are essential to plasma cell differentiation in inflamed tissue dependent on IL-1R

Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.

CD28 Induces Mitochondrial Respiration through Irf4 for Long Lived Plasma Cells Survival

Successful vaccination strategies against emerging pathogens like that of the Zika virus depend upon the sustained production of antigen-specific antibodies by terminally differentiated B cells known as plasma cells. Because the half-life of circulating antibodies is on the order of days to weeks, durable humoral immunity requires the induction of long-lived plasma cells (LLPCs) that home to specialized survival niches in the bone marrow and live indefinitely. Therefore, understanding the cellular and molecular interactions that govern LLPC survival are critical for the design of effective vaccines.We have published that CD28, the canonical T cell co-stimulatory molecule, is required for LLPC survival in the bone marrow. In order to maintain this extraordinary degree of longevity, LLPCs must be able to compete successfully in an environment wherein normal hematopoiesis puts constraints on the availability of nutrients. Furthermore, LLPCs are highly biosynthetic as antibody producing factories and necessitate a high level of metabolic activity. In T cells CD28 is known to induce glycolysis at the expense of mitochondrial respiration. To our great surprise, CD28 signaling in LLPCs increases mitochondrial respiration whilst not greatly affecting glycolysis. However, CD28 enhanced the glycolytic capacity and reserve, suggesting that CD28 signaling regulates their ability to better compete for nutrient availability and metabolic fitness. CD28 induces the expression of the glucose transporter Glut1 and subsequent glucose uptake as assessed by staining with the autofluorescent glucose analogue NBDG. In contrast to the T cell literature, CD28 increases the mitochondrial mass and membrane potential in LLPCs, suggesting that rather than using glucose primarily for glycolysis it is converted into pyruvate which then is used in mitochondrial respiration.Mechanistically, CD28 induces NFkB dependent IRf4 upregulation, a transcription factor known to regulate metabolism in T cells and survival in LLPCs. Furthermore, inhibition of NFkB abrogates CD28-induced increases in glucose uptake and mitochondrial mass. We have recently published that the Grb2/Vav binding domain on the CD28 cytoplasmic tail is required for CD28-mediated survival. In mice wherein this domain is mutated (AYAA mutants), LLPCs have decreased glucose uptake and mitochondrial mass. Plasma cells from AYAA mutant mice also have decreased levels of Irf4, facilitating a model wherein CD28 through its Grb2/Vav binding domain increased Irf4 for metabolic fitness. We also describe for the first time an NFkB superenhancer element upstream of the Irf4 transcriptional start site, suggesting that CD28 may govern both direct Irf4 promoter activity as well as DNA folding for increased Irf4 production. By using siRNA to target Irf4 at the genetic level, we see diminished mitochondrial mass, suggesting that Irf4 is the direct regulator of CD28-regulated metabolism in LLPCs.A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). CD28 increased the amount of ROS production in LLPCs as seen by staining with a mitochondrial-ROS specific dye. This is counterintuitive, as ROS are well characterized cell damaging agents. Paradoxically, when we inhibited ROS with MnTBAP, a ROS scavenger, CD28 no longer conferred a survival signal in LLPCs. Taken together these results suggest a model wherein CD28 through its Grb2/Vav binding domain induces NFkB dependent upregulation of Irf4 directly on the promoter and augments further Irf4 production via a previously undescribed superenhancer element upstream of the Irf4 gene. Irf4 then goes on to increase mitochondrial respiration for CD28-mediated LLPC survival and metabolic fitness. This understanding will not only allow for the augmentation of vaccine design and efficacy, but may also lead to novel therapeutic strategies to combat antibody-mediated autoimmunity. DisclosuresNo relevant conflicts of interest to declare.

CD28 induces mitochondrial respiration through Slp-76 in Long-Lived Plasma Cells for ROS-dependent survival.

Abstract Sustained humoral immunity is dependent upon bone marrow (BM) resident long-lived plasma cells (LLPCs). We have shown that CD28 signaling in LLPCs is necessary for their survival and antibody production; however, the mechanistic basis for this is unclear. In T cells, CD28 induces glycolysis at the expense of mitochondrial respiration. To our great surprise, CD28 induces mitochondrial respiration and increases the spare respiratory capacity in LLPCs, but not in short lived plasma cells (SLPCs), through Slp-76. While not affecting glycolysis, CD28 does increase the glycolytic capacity and glycolytic reserve specifically in LLPCs. Paradoxically, the mitochondrial respiration dependent production of reactive oxygen species (ROS) is required for CD28 mediated survival. Mechanistically, ROS dependent activation of NFkB drives Irf4 expression, which itself upregulates myc, both of which have been described as central regulators of metabolism in T cells. In addition, CD28 drives myc-dependent mitochondrial biogenesis in LLPCs, thereby sustaining mitochondrial respiration-dependent ROS. This leads to a self-reinforcing model wherein CD28 induces mitochondrial respiration for ROS-dependent activation of Irf4 and myc, which then drive further mitochondrial biogenesis and enhanced metabolic fitness for LLPC survival. Our work suggests that CD28, by increasing metabolic fitness, may provide a qualitatively distinct program of longevity that allows greater competitive advantage for survival in the BM microenvironment. CD28 may therefore regulate LLPC induction, survival, and function, making it an attractive target for the augmentation of vaccine design as well as in the alleviation of antibody mediated autoimmunity.