Megan Murray

Novel regulation of CD80/CD86-induced phosphatidylinositol 3-kinase signaling by NOTCH1 protein in interleukin-6 and indoleamine 2,3-dioxygenase production by dendritic cells.

Background: Engagement of CD80/CD86 on dendritic cells by CD28 on T cells induces dendritic cell production of IL-6 and IDO. Results: The NOTCH pathway modulates activation of the PI3K pathway downstream of CD80/CD86 ligation and regulates IL-6 and IDO production. Conclusion: Cross-talk between NOTCH and PI3K pathways modulates dendritic cell production of IL-6 and IDO. Significance: Elucidating the molecular mechanism of NOTCH-PI3K cross-talk will have broad implications in human disease. Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement of the prototypic T cell costimulatory receptor CD28 by the cognate ligands CD80/CD86, expressed on DC. Although CD28 signaling in T cell activation has been well characterized, it has only recently been shown that CD80/CD86, which have no demonstrated binding domains for signaling proteins in their cytoplasmic tails, nonetheless also transduce signals to the DC. Functionally, CD80/CD86 engagement results in DC production of the pro-inflammatory cytokine IL-6, which is necessary for full T cell activation. However, ligation of CD80/CD86 by CTLA4 also induces DC production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which depletes local pools of the essential amino acid tryptophan, resulting in blockade of T cell activation. Despite the significant role of CD80/CD86 in immunological processes and the seemingly opposing roles they play by producing IL-6 and IDO upon their activation, how CD80/CD86 signal remains poorly understood. We have now found that cross-linking CD80/CD86 in human DC activates the PI3K/AKT pathway. This results in phosphorylation/inactivation of its downstream target, FOXO3A, and alleviates FOXO3A-mediated suppression of IL-6 expression. A second event downstream of AKT phosphorylation is activation of the canonical NF-κB pathway, which induces IL-6 expression. In addition to these downstream pathways, we unexpectedly found that CD80/CD86-induced PI3K signaling is regulated by previously unrecognized cross-talk with NOTCH1 signaling. This cross-talk is facilitated by NOTCH-mediated up-regulation of the expression of prolyl isomerase PIN1, which in turn increases enzyme activity of casein kinase II. Subsequently, phosphatase and tensin homolog (which suppresses PI3K activity) is inactivated via phosphorylation by casein kinase II. This results in full activation of PI3K signaling upon cross-linking CD80/CD86. Similar to IL-6, we have found that CD80/CD86-induced IDO production by DC at late time points is also dependent upon the PI3K → AKT → NF-κB pathway and requires cross-talk with NOTCH signaling. These data further suggest that the same signaling pathways downstream of DC CD80/CD86 cross-linking induce early IL-6 production to enhance T cell activation, followed by later IDO production to self-limit this activation. In addition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novel cross-talk between NOTCH1 and PI3K signaling may provide new insights in other biological processes where PI3K signaling plays a major role.

CD28-mediated pro-survival signaling induces chemotherapeutic resistance in multiple myeloma

Chemotherapeutic resistance remains a significant hurdle in the treatment of multiple myeloma (MM) and is significantly mediated by interactions between MM cells and stromal cells of the bone marrow microenvironment. Despite the importance of these interactions, the specific molecules and downstream signaling components involved remain incompletely understood. We have previously shown that the prototypic T-cell costimulatory receptor CD28, which is also expressed on MM cells, is a key mediator of MM survival and apoptotic resistance. Crosslinking CD28 by agonistic antibodies or myeloid dendritic cells (DC; these express the CD28 ligands CD80/CD86) prevents apoptosis caused by chemotherapy or serum withdrawal. We now report that CD28 pro-survival signaling is dependent upon downstream activation of phosphatidyl-inositol 3-kinase/Akt, inactivation of the transcription factor FoxO3a, and decreased expression of the pro-apoptotic molecule Bim. Conversely, blocking the CD28-CD80/CD86 interaction between MM cells and DC in vitro abrogates the DCs ability to protect MM cells against chemotherapy-induced death. Consistent with these observations, in vivo blockade of CD28-CD80/CD86 in the Vk*MYC murine myeloma model sensitizes MM cells to chemotherapy and significantly reduces tumor burden. Taken together, our findings suggest that CD28 is an important mediator of MM survival during stress and can be targeted to overcome chemotherapy resistance.

CD28 Promotes Plasma Cell Survival, Sustained Antibody Responses, and BLIMP-1 Upregulation through Its Distal PYAP Proline Motif

In health, long-lived plasma cells (LLPC) are essential for durable protective humoral immunity, and, conversely, in disease are a major source of pathogenic Abs 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 Ab titers, by supporting the survival of LLPC, but not short-lived PC (SLPC). We now find that, unlike SLPC, CD28 activation in LLPC induces prosurvival downstream Vav signaling. Knockin mice with CD28 cytoplasmic tail mutations that abrogate Vav signaling (CD28-AYAA) had significantly fewer LLPC but unaffected SLPC numbers, whereas mice with mutations that abrogate PI3K signaling (CD28-Y170F) were indistinguishable from wild-type controls. This was consistent with the loss of CD28’s prosurvival 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 Ag-specific LLPC populations and Ab titers in vivo. Signaling downstream of the CD28 Vav motif induced previously undescribed transcriptional regulation of B lymphocyte–induced maturation protein-1, a key mediator of PC differentiation and maintenance. These findings suggest CD28 signaling in LLPC modulates the central B lymphocyte–induced maturation protein-1 transcriptional nexus involved in long-term survival and function.