Sustained Antibody Responses Depend on CD28 Function in Bone Marrow Resident Plasma Cells

Abstract

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.

Plasma cells: You are what you eat

“…23 Genes such as BLIMP-1, Myeloid cell leukemia 1 (MCL1), CD28, and B-cell maturation antigen (BCMA), which are essential for the long-term survival of plasma cells, were similarly expressed transcriptionally across all mature mouse plasma cells irrespective of their longevity. 1,23,105,[132][133][134][135] In previous work, we and others demonstrated that the transcription factor ZBTB20 promotes plasma cell longevity. 136,137 Reciprocally, ZBTB32, a related family member, antagonizes plasma cell longevity.…”

“…132,133 A number of other extrinsic stimuli, such as IL-6 and Tumor Necrosis Factor α, and engagement of CD28 and CD44 also promote plasma cell survival ex vivo. 134,177,178 Genetic knockout studies, however, have provided mixed results, suggesting that plasma cell-intrinsic signaling by these factors might not all be essential in vivo. 133,179 A number of accessory cell types such as eosinophils, basophils, megakaryocytes, regulatory T cells, and mesenchymal cells have all been proposed to enhance plasma cell survival.…”

CD19 as a therapeutic target in a spontaneous autoimmune polyneuropathy

“…There is also evidence for the intrinsic function of CD28 in the survival of plasma cells [24]. Loss of CD28 or its ligands B7-1 and B7-2 results in altered frequency of plasma cells and antibody levels, although debate remains as to whether CD28 is a positive or negative regulator of plasma cells [25,26]. In this study, we provide evidence that B cells contribute to the pathogenesis of SAP in B7-2 KO NOD mice, irrespective of the complex sequelae of B7-2 elimination on humoral immunity.…”

Differentiation and maintenance of long-lived plasma cells

“…CD28 deficiency in the B cell compartment only affects the maintenance of plasma cell in the bone marrow. The threshold for CD28-dependent NF-B activation is much lower in bone marrow plasma cells compared to splenic plasma cells [42]. Because CD80 (a ligand for CD28) is expressed on CXCL12 hi reticular cells, bone marrow plasma cells might obtain significant survival advantage by their interaction with the CXCL12 hi reticular cells.…”