Role of CD28-mediated signaling in multiple myeloma

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CD28 is a key co-stimulatory molecule expressed by naive and activated T cells, but is also found on short- and long-lived primary plasma cells (PC) and on mouse and human multiple myeloma (MM) cells, where its expression correlates with disease progression. Most published data suggest a role of CD28 in cell survival and sustained immunoglobulin (Ig) production, while others describe opposite effects.We found that ligation of CD28 with the superagonistic antibody TGN1412 was often not sufficient for activating signals (p-ERK, p-Akt, NFκB) in MM cells but required sub-signaling traces of phorbol-dibutyrate, suggesting that CD28 signaling depends on additional interactions with neighboring PC or other cells in BM niches.We have generated a mouse strain confining inactivation of CD28 to the B cell lineage by introducing an mb1Cre transgene into mice with a partially “floxed” CD28 gene. First data suggest diminished IgM and IgG production upon loss of CD28. However, no complete deletion of CD28 was achieved. To overcome this, we currently include breedings with CD19Cre, CD23Cre and AIDCre. We had also proposed to use B cell-specific CD28-deficient mice in a model of spontaneous MM. This had to be abandoned because compatibility with the use of Cre recombinase could not be established. We now turned to another model (iMyc mice), in which plasmacytoma are induced by pristane. We are currently crossing these mice to CD28fl/fl x mb1Cre mice. Here, incomplete deletion of CD28 will show if plasmacytoma preferably develop or expand when CD28 is present or not. In the meantime, we have exploited our established model of B lineage-specific ablation of the transcription factor C/EBPβ which shows impaired development of PC both in vitro and in vivo. Our results suggest that the two larger CEBPβ isoforms LAP* and LAP support survival and proliferation of human MM cell lines.For the next funding period, we propose to identify the putative natural co-factor required for effective CD28 signaling and the cell(s) expressing it. Likely co-stimuli provided by the interacting cells will be inhibited by blocking antibodies or mimicked by agonistic antibodies. A comprehensive analysis of the responding genes, will be performed by cDNA microarray analysis. With regard to in vivo studies, we will now apply the new inducible myeloma mouse model with lineage-specific and inducible ablation of CD28 expression to obtain answers about the role of CD28 for MM-development, survival and function. In addition, we will further dissect the role of B cell lineage-expressed CD28 (with different cre-deleters) for the humoral immune response and clarify the discrepancies with some reports from the literature.If CD28 is beneficial for MM survival, blocking antibodies which do not interfere with T cell activation could be a therapeutic approach. We will develop an antibody which recognizes CD28 with one arm and CD138 with the other one. If affinity is greatly boosted by dual binding, this bi-specific antibody would be a good candidate to test in our model systems.