Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c− plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c− IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c− precursors (>7-fold). Freshly sorted CD11c+ but not CD11c− cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c− cells can be induced to secrete high levels of IFN-α, in response to influenza virus. CD11c+ and CD11c− cells can mature in vitro with GM-CSF + TNF-α or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c− subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.

SUMMARYNicotine alters a wide range of immunological functions, including innate and adaptive immune responses. To date, no studies have been reported showing the immunoregulatory effects of nicotine on dendritic cells (DCs), which are critical cells for initiation of cell-mediated immunity against infection and neoplastic diseases. In this work, we report that, in a nicotinic environment, monocyte-derived DCs manifest lower endocytic and phagocytic activities. Interestingly, although immature DCs undergo maturation in response to bacterial antigen lipopolysaccharide, they produce decreased levels of pro-in¯ammatory cytokines, notably interleukin-12, and reveal a reduced ability to stimulate antigen-presenting cell-dependent T-cell responses. Importantly, the reduction in T-cell responses is associated with a diminished ability of DCs to induce differentiation and expansion of type 1 T cells, as evidenced by a decreased frequency of interferon-g-producing effector cells. These results strongly suggest that nicotine can exert its immunosuppressive effects on immune surveillance through functional impairment of the DC system.

T cell subsets differ in their metabolic requirements, and further insight into such differences might be harnessed to selectively promote regulatory T cells (Tregs) for therapies in autoimmunity and transplantation. We found that Gln restriction during human T cell activation favored CD4 T cells with high expression of the Treg transcription factor FOXP3. This resulted from shrinking numbers and reduced proliferation of activated FOXP3lo/−CD4 T cells while FOXP3hiCD4 T cell numbers increased. This gain was abolished by blocking Gln synthetase, an enzyme that responds to Gln and purine/pyrimidine deficiencies. The shift toward FOXP3hiCD4 T cells under Gln restriction was recapitulated with inhibitors of Gln-dependent pyrimidine and purine syntheses that together closely mimicked declining cell numbers and cell cycles, and by small interfering RNA knockdown of the respective rate-limiting Gln-consuming enzymes CAD and PPAT. FOXP3hi-enriched CD25hiCD4 T cells from these cultures inhibited proliferation, but they also produced effector cytokines, including IL-17A. The latter was largely confined to CTLA-4hi-expressing FOXP3hi-enriched CD25hiCD4 T cells that suppressed proliferation more weakly than did CTLA-4lo/−CD25hiFOXP3hi–enriched T cells. A causal link between high IL-17A production and impaired suppression of proliferation could not be demonstrated, however. Collectively, these results reveal a Gln synthetase–dependent increase and resilience of FOXP3hi cells under Gln restriction, and they demonstrate that impaired Gln-dependent nucleotide synthesis promotes FOXP3hi cells with regulator properties. It remains to be investigated to what extent the concomitant retention of IL-17A–producing CD4 T cells may limit the therapeutic potential of Tregs enriched through targeting these pathways in vivo.

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