Dendritic Cell (DC) differentiation is a complex system involving multiple progenitors with potential to differentiate into a variety of DC subsets. Understanding the mechanisms regulating these differentiation pathways is critical to understanding how defective DCs arise in cancer. Impaired DC differentiation often results in immunosuppressive cells that either hinder immune activation in disease or promote tumor growth and metastasis. We previously established that the serine-threonine kinase Protein Kinase C β isoform II (PKCβII) is required for human DC differentiation from CD34+ progenitor cells and monocytes, and have recently found that murine bone marrow (BM) cells also need it to become fully differentiated and functional DCs. However, the molecular targets of PKCβII in this pathway remain unclear. It is well established that the transcription factors Interferon Regulatory Factors 4 and 8 (IRF4 and IRF8) are also important for DC differentiation. IRF4 is crucial for the development of conventional DCs (mediated by GM-CSF), while IRF8 is crucial for the development of plasmacytoid and CD8α+DCs (mediated by FLT3-L). We hypothesized that a relationship existed between PKCβII and IRF4/8, and investigated the effects of PKC activation on IRF4/8 expression.
Using human progenitor cell lines and murine BM cells we found that PKC activation upregulated IRF4 and IRF8 expression, while PKC inhibition downregulated IRF4 and IRF8. PKC inhibition also prevented these cells from differentiating into DCs, as determined by their phenotypic markers, physical characteristics, and T-cell stimulatory activity. However, we found that in progenitor cells GM-CSF (a known PKCβII activator) decreased IRF8 expression while upregulating IRF4 expression. This led us to investigate the differential effects of GM-CSF and FLT3-L on the PKC-IRF relationship. We saw that FLT3-L treatment of murine BM cells caused an upregulation of IRF8 and stimulated DC differentiation, and that DC differentiation and IRF8 upregulation were both lost in the presence of a PKC inhibitor. Using Image Stream analysis we found that FLT3-L treatment of progenitor cells activated PKCβII and PKCα. To determine which PKC(s) mediates the FLT3-L driven upregulation of IRF8, we used PKCα knockout (KO) BM and saw that cells were still able to differentiate into DCs and IRF8 levels were still being upregulated. Thus, PKCβII is the PKC that mediates FLT3-L driven DC differentiation and IRF8 upregulation. To determine what molecules could be acting downstream of PKCβII in regulating IRF4/8, we again used Image Stream analysis and visualized STAT3 and STAT5 translocation into the nucleus. Using murine BM cells we found that STAT3 and IRF8 nuclear localization increased with FLT3-L treatment, while GM-CSF treatment caused increased STAT5 and IRF4 nuclear localization. When looking at human monocytes and the human monocytic progenitor cell lineTHP-1 we saw similar effects: GM-CSF treatment increased STAT5 and IRF4 nuclear localization, while pan-PKC inhibition decreased basal STAT5 and IRF4 nuclear localization. Interestingly, these human monocytes and THP-1 cells had lower nuclear levels of STAT3 and IRF8 following FLT3-L treatment – possibly because these cells are already somewhat committed to the monocyte-derived conventional DC pathway. However, in murine early progenitor cells, after 15 minutes of PKC activation we saw increased STAT3 activation, indicating that PKC-regulated STAT3 activation is playing a role earlier in the differentiation process. To find the progenitor cells immediately effected by PKCβII activation, we used IRF8-eGFP murine BM and saw that PKC activation caused induced IRF8 expression as early as in the multi-potent progenitor cells (MPP2 and MPP3), and this upregulation continued to increase as cells differentiated to CD11b+progenitor cells and GMP.
These studies indicate that PKCβII is activated in progenitor cells by either FLT3-L or GM-CSF, causing an upregulation of IRF8 or IRF4, respectively. PKCβII may be acting through STAT5 and STAT3 to induce IRF4 and IRF8, depending on the cytokine treatment. By having a better understanding of how PKCβII regulates the expression of these transcription factors, which are required for DC differentiation, we can manipulate the PKCβII-IRF relationship to drive or impair DC differentiation in pathological settings, and may improve DC-vaccine development.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.