The Role of CD28 in Long-lived Bone Marrow-Resident Plasma Cell Survival and Function
Rozanski, Cheryl Helene
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Durable protective immunity against infection requires sustained continuous antibody production by plasma cells (PC) for years to decades. It has been hypothesized that this is primarily the function of long-lived bone marrow resident PC (also called long-lived plasma cells (LLPC)) that survive for years to decades within specialized niches. What regulates/supports this survival and whether these LLPC are an intrinsically distinct subset of PC remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on both soluble factors and cell-to-cell interactions within the bone marrow microenvironment. We have shown that the prototypic T cell costimulatory receptor CD28 is expressed on normal and transformed LLPC, and plays an essential role in their survival. While normal LLPC and splenic short-lived plasma cells (SLPC) both expressed CD28, its activation in vitro only significantly increased survival and IgG production in LLPC. This was due to the ability of LLPC to signal through CD28, while the SLPC could not. Consistent with these findings in vitro , we have shown in vivo that bone marrow chimeras with a specific loss of CD28 in the B cell lineage had significantly reduced long-term antigen-specific antibody titers and decreased LLPC numbers (but not SLPC numbers). We believe these findings are the first to demonstrate the existence of an intrinsically distinct bone marrow (BM) LLPC subset necessary to sustain durable antigen specific antibody titers, and establish a central role for CD28 function in the maintenance of plasma cell survival and humoral immunity. As these data have shown CD28 signaling plays an important role in maintaining long-term humoral immune responses, we have begun to examine the mechanisms by which CD28 signaling affects PC function. To further elucidate CD28 signaling in BM PC, we utilized CD28 knock-in mice where the CD28 cytoplasmic tail was mutated at either the YMNM or proline-rich (PYAPP) motifs, resulting in loss of downstream PI3K or vav signaling respectively in T cells. We found CD28 activation in BM PC also induced downstream PI3K and vav signaling. Similar to what has been reported for T cells, we have found that CD28 activation increased activation of Akt in BM PC, which mediated increased expression of the glucose transporter Glut-1 and enhanced the survival of J558 cells (CD28 + transformed murine PC line) in glucose-limited conditions. These data suggest PI3K signaling downstream of CD28 activation is playing a role in regulating BM PC metabolic fitness. Relevant to the downstream vav pathway, recent studies have implicated a role for vav signaling in the transcriptional regulation of Blimp-1 expression, thought to be the "master" regulator of PC identity. We have found that in the CD28-vav signaling deficient mice the BM PC were selectively lost in vivo (with intact splenic PC populations) and BM PC survival could not be rescued in vitro by CD28 activation in serum starvation conditions. Surprisingly, BM PC survival was not affected by loss of downstream PI3K signaling. Furthermore, CD28 activation induced Prdm1 (the gene that encodes Blimp-1) transcription, which increased Blimp-1 protein expression via downstream vav signaling and increased promoter activity in BM PC. These findings suggest that CD28-vav signaling is transcriptionally regulating Blimp-1 expression. In T cells CD28 activation upregulates IL-2 transcription through a CD28RE composite element next to an AP-1 site within the IL-2 promoter, and similarly upregulates IL-8 expression via CD28RE site within the IL-8 promoter in myeloma cells. In silico analysis of the Prdm1 promoter revealed the presence of a previously undescribed CD28RE site, and by using site-directed mutagenesis studies it was determined to be required for CD28-mediated Prdm1 upregulation in the CD28 + J558 plasmacytoma cells. Furthermore, PC that had upregulated Prdm1 promoter activity following CD28 activation also upregulated expression of the pro-survival receptor B-cell maturation antigen (BCMA), suggesting an additional mechanism by which CD28 activation may support PC survival. Taken together, our data suggests the CD28-vav signaling pathway in PC induced a transcription factor that binds to the CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC survival and humoral immunity. In summary our findings suggest a model for CD28 in LLPC survival and function. We have shown that PC CD28 signaling is a defining intrinsic molecular component of LLPC and SLPC subsets, allowing the LLPC to access and utilize the limited number of prosurvival niches in the bone marrow. Furthermore our findings suggest CD28 downstream signaling separately regulates BM PC survival through the vav pathway (via induction of Blimp-1) and BM PC metabolic fitness through the PI3K pathway. These findings suggest PC CD28 might play a critical role in attenuating LLPC survival in autoimmunity, multiple myeloma, and organ graft rejection. Therapeutically targeting CD28 might selectively inhibit auto-reactive and/or pathogenic LLPC in human disease.