Retinoic acid induction of CD1d expression primes chronic lymphocytic leukemia B cells for killing by CD8+ invariant natural killer T cells.

Invariant natural killer T (iNKT) cells are cytotoxic T cells that respond to glycolipid antigens presented by CD1d. Therapeutic activation of iNKT cells with α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in mice and clinical trials involving α-GalCer-stimulated iNKT cells are ongoing in humans. B cells express CD1d, however, we show that CD1d expression is reduced on B cells from patients with chronic lymphocytic leukemia (CLL). B cells from CLL patients pulsed with α-GalCer failed to stimulate cytolytic degranulation by iNKT cell lines, but could present the more potent glycolipid analogue, 7DW8-5. Retinoic acid receptor-α (RAR-α) agonists induced CD1d expression by CLL B cells, restoring their ability to present α-GalCer to CD8α+ iNKT cells, resulting in cytolytic degranulation. Thus, RAR-α agonists can augment the anti-tumor activities of iNKT cells against CLL cells in vitro. Their inclusion in iNKT cell-based therapies may benefit patients with CLL.

Design, synthesis, and functional activity of labeled CD1d glycolipid agonists.

Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR-α-GalCer-CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR-glycolipid-CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists.

CD1d blockade suppresses the capacity of immature dendritic cells to prime allogeneic T cell response.

BACKGROUND: Dendritic cells (DCs) are the principal antigen-presenting cells involved in primary immune response and immunoregulation. The function of DCs is believed to depend on their degree of maturation. Mature DCs activate immune responses, whereas immature DCs (imDCs) tend to induce immune tolerance. CD1 is involved in regulating the development of imDCs, which have important roles in initiating or suppressing the immune response after transplantation. MATERIALS AND METHODS: We used male BALB/c mice and C57BL/6 mice (aged 8-10 wk, 18-22 g). We isolated and purified T lymphocytes from mouse spleen. Immature DCs modified by viral delivery of interleukin-10 (IL-10) were stimulated with granulocyte macrophage colony-stimulating factor and lipopolysaccharide (LPS) and treated with anti-CD1d in vitro. We used mixed lymphocyte cultures to evaluate the heterogeneity of T lymphocyte response. We also examined the proliferation of T lymphocytes and the expression of cytokines. RESULTS: CD1d blockade did not impair granulocyte macrophage colony-stimulating factor and LPS-stimulated DC maturation. We observed a dramatic increase in allogeneic T lymphocyte proliferation (stimulation index) at all tested responder-stimulator ratios in response to imDCs cultured in the presence of LPS (P < 0.05). CD1d has an important role in imDC-primed T cell response (P < 0.05). CD1d blockade reduced the capacity of imDCs to prime allogeneic T cells. T cells pre-sensitized by LPS-stimulated imDCs showed remarkably elevated proliferation in response to T cells from either BALB/c or C57BL/6 mice (P < 0.01). We observed a significant decrease in the proliferation of T cells pre-sensitized by stimulated imDCs after CD1d blockade. Lipopolysaccharide stimulation caused elevated the production of IL-12 and tumor necrosis factor-α (TNF-α) (P < 0.01) and decreased the secretion of IL-10 (P < 0.05). The addition of CD1d neutralization antibody did not significantly change the concentrations of IL-12, TNF-α, or IL-10 produced by imDCs cultured in the presence of LPS (P > 0.05). CONCLUSIONS: Blockade of CD1d impaired the ability of imDCs to stimulate allogeneic T cell response. By reduced T cell proliferation, the secretion of IL-12 and TNF-α decreased and production of a T-helper type 2 cytokine IL-10 increased, which indicates the potential of CD1d blockade as a method to induce immune tolerance to allograft antigens in transplantation.

Antigen-induced increases in pulmonary mast cell progenitor numbers depend on IL-9 and CD1d-restricted NKT cells.

Pulmonary mast cell progenitor (MCp) numbers increase dramatically in sensitized and aerosolized Ag-challenged mice. This increase depends on CD4(+) T cells, as no MCp increase occurs in the lungs of sensitized wild-type (WT) mice after mAb depletion of CD4(+) but not CD8(+) cells before aerosol Ag challenge. Neither the genetic absence of IL-4, IL-4Ralpha chain, STAT-6, IFN-gamma, or IL-12p40 nor mAb blockade of IFN-gamma, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12p40, or IL-12p40Rbeta1 before Ag challenge in WT mice reduces the pulmonary MCp increase. However, sensitized and Ag-challenged IL-9-deficient mice and sensitized WT mice given mAb to IL-9 just before Ag challenge show significant reductions in elicited lung MCp/10(6) mononuclear cells of 47 and 66%, respectively. CD1d-deficient mice and WT mice receiving anti-CD1d before Ag challenge also show significant reductions of 65 and 59%, respectively, in elicited lung MCp/10(6) mononuclear cells, revealing an additional requirement for MCp recruitment. However, in Jalpha18-deficient mice, which lack only type 1 or invariant NKT cells, the increase in the numbers of lung MCp with Ag challenge was intact, indicating that their recruitment must be mediated by type 2 NKT cells. Furthermore, anti-CD1d treatment of IL-9-deficient mice or anti-IL-9 treatment of CD1d-deficient mice does not further reduce the significant partial impairment of MCp recruitment occurring with a single deficiency. These findings implicate type 2 NKT cells and IL-9 as central regulators that function in the same pathway mediating the Ag-induced increase in numbers of pulmonary MCp.