CD28 signaling is essential for maintenance of long-term antigen-specific antibody production and for persistence of plasma cells in the bone marrow of mice.

Interactions between the malignant plasma cells of multiple myeloma (MM) and stromal cells within the bone marrow (BM) microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal BM-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce pro-survival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor prognosis subgroups, and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell (DC) directly transduces a pro-survival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal DC to produce the pro-survival cytokine IL-6 (involving novel crosstalk with the Notch pathway) and the immunosuppressive enzyme indoleamine 2, 3 dioxygenase (IDO). These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, and point to similar interaction for normal plasma cells as well as suggesting novel therapeutic strategies to target malignant and pathogenic (e.g. in allergy and autoimmunity) plasma cells.

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.

In health, long-lived plasma cells (LLPC) are essential for durable protective humoral immunity, and conversely in disease are a major source of pathogenic antibodies in autoimmunity, graft rejection and allergy. However, the molecular basis for their longevity is largely unknown. We have recently found that CD28 signaling in plasma cells (PC) is essential for sustaining antibody titers, by supporting the survival of LLPC but not short-lived PC (SLPC). We now find that unlike SLPC, CD28 activation in LLPC induces pro-survival downstream Vav signaling. Knock-in mice with CD28 cytoplasmic tail mutations that abrogate Vav signaling (CD28-AYAA) had significantly fewer LLPC but unaffected SLPC numbers, while mice with mutations that abrogate PI3K signaling (CD28-Y170F) were indistinguishable from WT controls. This was consistent with the loss of CD28’s pro-survival effect in LLPC from CD28-AYAA, but not CD28-Y170F, mice. Furthermore, the CD28 Vav motif in the B lineage was essential for the long-term maintenance of antigen-specific LLPC populations and antibody titers in vivo. Signaling downstream of the CD28 Vav motif induced previously undescribed transcriptional regulation of BLIMP-1, a key mediator of PC differentiation and maintenance. These findings suggest CD28 signaling in LLPC modulates the central BLIMP-1 transcriptional nexus involved in long-term survival and function.

SUMMARY Durable 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.

Our recently published data demonstrate significant similarities between normal and malignant plasma cells in the cellular and molecular interactions that support their survival in the bone marrow microenvironment, and suggest that the biology of multiple myeloma may largely reflect that of their normal counterparts.

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<0.05), whereas CD28 activation of SLPC did not induce survival. Addition of BMDC improved the survival of LLPC 2-fold over that seen with media alone, and resulted in a significant increase in IgG production (p<0.001). In contrast, CD28-/- PC had no increase in survival when cocultured with BMDC, suggesting a direct role for CD28 in PC-DC interaction. Consistent with these findings we now show that in vivo, vaccinated bone marrow CD28-/-:μMT chimeras had significantly reduces long-term antibody titers and LLPC (but not SLPC) survival from t1/2 of 426 to 63 days. Additionally, LLPC CD28 modulates the microenvironment by inducing CD80/CD86+ stromal cell production of the supportive cytokine IL-6 (p<0.001 vs. BMDC/PC alone), which was abrogated by blocking CD80 and CD86 (p<0.05). From the above experiments we hypothesized IL-6 was playing a significant role in the survival of LLPC, however to our surprise LLPC cocultured with WT or IL-6-/- BMDC maintained equivalent LLPC numbers, interestingly however LLPC cocultures with BMDC showed a 3-fold increase of IgG compared to LLPC cocultured with IL-6-/- BMDC (p<0.001). These data suggest CD28 is a key molecular component in LLPC survival, whereas IL-6 contributes to Ig production. Our data demonstrates that signaling through CD28 directly supports the survival of LLPC, sustaining long term protective antibody titers. These findings suggest CD28 plays an important role in maintaining the quality of protective durable humoral immunity. Strategies to augment CD28 signaling may lead to greater LLPC survival and persistent antibody titers in cancer vaccine development. Conversely, blocking CD28 signaling could compromise the survival of transformed myeloma cells which are critically dependent on the bone marrow microenvironment. Disclosures: Boise: University of Chicago: Patents & Royalties.

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.