Yuekang Xu

Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.

To gain ample numbers of dendritic cells (DCs) for investigation, or for immunotherapy, the culture of DC precursors from bone marrow in either GM-CSF and IL-4 (GM/IL4-DCs) or Flt3L (FL-DCs) has often been used. Despite their common use, the relationship of these culture-derived DCs to those in vivo, and their relative potential for use in immunotherapy, needs further elucidation. In this study we found that in contrast to FL-DCs, highly purified GM/IL4-DCs were larger and more granular, surface Mac-3+, and were comprised of two populations (CD24lowCD11bhigh and CD24highCD11blow). Functionally, although comparable in T cell activation, GM/IL4-DCs produced more inflammatory mediators including TNF-α, IL-10, CCL-2, and NO than FL-DCs upon TLR ligation. However, FL-DCs migrated more efficiently to draining lymph nodes after s.c. injection and produced a different profile of cytokines to GM/IL4-DCs. Developmentally, unlike GM/IL4-DCs, FL-DCs cannot be differentiated from CD11bhighLy6ChighLy6G− monocytes. Collectively, these data suggest that the GM/IL4-DCs are the equivalents of the TNF-α and inducible NO synthase producing DCs in vivo that emerge after inflammation whereas FL-DCs better represent the steady-state resident DCs. The differences between GM/IL4-DCs and FL-DCs have serious implications for DC-based immunotherapeutic strategies.

Loss of the Pro-Apoptotic BH3-only Bcl-2 Family Member Bim Inhibits BCR Stimulation–induced Apoptosis and Deletion of Autoreactive B Cells

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.

GM-CSF increases cross-presentation and CD103 expression by mouse CD8⁺ spleen dendritic cells.

Resident CD8+ DCs perform several functions, including cross‐presenting antigen and rapidly engulfing the Gram‐positive intracellular pathogen Listeria monocytogenes. Little is known about how these functions of CD8+ DCs are modulated. Here, we show that granulocyte‐macrophage CSF (GM‐CSF), a cytokine that exists at low levels at steady state but is elevated during infection and inflammation, enhances cross‐presentation and rapid uptake of L. monocytogenes by resident CD8+ DCs. This previously unrecognized functional enhancement of CD8+ DCs by GM‐CSF was independent of promoting DC survival in vitro. Enhancement of these functions by GM‐CSF was also marked by CD103 expression on CD8+ DCs that was strongly regulated by GM‐CSF. Our findings not only identify GM‐CSF as a key molecule regulating CD8+ DC function, but also as a factor responsible for functional heterogeneity of CD8+ DCs that is at least substantially demarcated by CD103 expression.

A requirement for CD45 distinguishes Ly49D-mediated cytokine and chemokine production from killing in primary natural killer cells

Engagement of receptors on the surface of natural killer (NK) cells initiates a biochemical cascade ultimately triggering cytokine production and cytotoxicity, although the interrelationship between these two outcomes is currently unclear. In this study we investigate the role of the cell surface phosphatase CD45 in NK cell development and intracellular signaling from activating receptors. Stimulation via the major histocompatibility complex I–binding receptor, Ly49D on CD45 −/− primary NK cells resulted in the activation of phosphoinositide-3-kinase and normal cytotoxicity but failed to elicit a range of cytokines and chemokines. This blockage is associated with impaired phosphorylation of Syk, Vav1, JNK, and p38, which mimics data obtained using inhibitors of the src-family kinases (SFK). These data, supported by analogous findings after CD16 and NKG2D stimulation of CD45 −/− primary NK cells, place CD45 upstream of SFK in NK cells after stimulation via immunoreceptor tyrosine-based activation motif-containing receptors. Thus we identify CD45 as a pivotal enzyme in eliciting a precise subset of NK cell responses.

The regulation of the development and function of dendritic cell subsets by GM-CSF: More than a hematopoietic growth factor

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that functions as a hematopoietic growth factor for the generation of white blood cells and is used clinically to stimulate hematopoiesis following chemotherapy. Apart from stimulating production of granulocytes and monocytes/macrophages, GM-CSF has also long been used for in vitro survival/generation of dendritic cells (DCs) from monocytes and bone marrow cells. Evidence has emerged pointing to an additional role for GM-CSF in regulating the function and differential development of several DC subsets. These newly ascribed functions of GM-CSF may underscore its importance in immunity against pathogens as well as initiating/mediating immunopathology in chronic inflammation. Here we summarize recent advances on the role of GM-CSF in regulating the development and function of DC subsets and discuss the biological significance of these new findings.

Resident and Monocyte-Derived Dendritic Cells Become Dominant IL-12 Producers under Different Conditions and Signaling Pathways

IL-12 is such a pivotal cytokine that it has been called the third signal for T cell activation, TCR engagement being the first and costimulation being the second. It has been generally viewed that the resident CD8+ dendritic cell (DC) subset is the predominant IL-12–producing cell type. In this study, we found, although this is so under steady state conditions, under inflammatory conditions monocyte-derived DC (mDC) became a major cell type producing IL-12. Depletion of either type of DC resulted in reduced production of IL-12 in vivo. For CD8+ DC, IL-12 production could be stimulated by various pathways viz. signaling through MyD88, Trif, or nucleotide-binding oligomerization domain (Nod)-like receptors. In contrast, for mDC, IL-12 production was mainly dependent on MyD88 signaling. Thus, conventional DCs and mDCs use different pathways to regulate IL-12 production.

The inflammatory cytokine, GM-CSF, alters the developmental outcome of murine dendritic cells

Fms‐like tyrosine kinase 3 ligand (Flt3L) is a major cytokine that drives development of dendritic cells (DCs) under steady state, whereas GM‐CSF becomes a prominent influence on differentiation during inflammation. The influence GM‐CSF exerts on Flt3L‐induced DC development has not been thoroughly examined. Here, we report that GM‐CSF alters Flt3L‐induced DC development. When BM cells were cultured with both Flt3L and GM‐CSF, few CD8+ equivalent DCs or plasmacytoid DCs developed compared to cultures supplemented with Flt3L alone. The disappearance of these two cell subsets in GM‐CSF + Flt3L culture was not a result of simple inhibition of their development, but a diversion of the original differentiation trajectory to form a new cell population. As a consequence, both DC progeny and their functions were altered. The effect of GM‐CSF on DC subset development was confirmed in vivo. First, the CD8+ DC numbers were increased under GM‐CSF deficiency (when either GM‐CSF or its receptor was ablated). Second, this population was decreased under GM‐CSF hyperexpression (by transgenesis or by Listeria infection). Our finding that GM‐CSF dominantly changes the regulation of DC development in vitro and in vivo has important implications for inflammatory diseases or GM‐CSF therapy.

Targeting IRF4 in autoimmune diseases.

As a member of the IRF family of transcription factors, interferon regulatory factor 4 (IRF4) is expressed in most cell types of the immune system. Recent findings suggest that IRF4 is essential for the development and function of T helper (Th) cell, regulatory T (Treg) cell, B cell, as well as dendritic cell (DC). Since these cells are crucial in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease (Crohns disease and ulcerative colitis), and type I diabetes, the roles of IRF4 in the initiation and progression of autoimmune diseases can not be neglected. In this review, we summarize recent advances on the pathological roles of IRF4 in autoimmunity and discuss the therapeutic significance of these new findings.

The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent.

The cell surface glycoprotein CD19 and the Src-related protein tyrosine kinase Lyn are key mediators of, respectively, positive and negative signaling in B cells. Despite the apparent opposition of their regulatory functions, a recent model of the biochemical events after B cell receptor (BCR) ligation intimately links the activation of Lyn and CD19. We examined the biochemical consequences of BCR ligation in mouse B cells lacking either Lyn or CD19 for evidence of interaction or codependence. In contrast to published results, we found CD19 phosphorylation after BCR ligation to be unaffected by the absence of Lyn, yet dependent on Src family protein tyrosine kinases as it was inhibited fully by PP2, an Src family-specific inhibitor. Consistent with normal CD19 phosphorylation in lyn−/− B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and extracellular signal-regulated kinase 1/2 activation after coligation with the BCRs were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. CD19−/− B cells were normal for increased Lyn kinase activity after BCR ligation, inhibition of BCR-mediated calcium flux after CD22 coligation, and inhibition of extracellular signal-regulated kinase phosporylation after FcγRIIB coligation. Collectively, these data show that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. We conclude that the roles of Lyn and CD19 after BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory.

IL-10 Controls Cystatin C Synthesis and Blood Concentration in Response to Inflammation through Regulation of IFN Regulatory Factor 8 Expression

Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid β associated with Alzheimer’s disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-β antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10–dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.