Vα14 TCR expressing invariant NK T (iNKT) cells recognize α-galactosylceramide (αGC)/CD1d complex and produce large amounts of various cytokines before the onset of the adaptive immunity. After stimulation with a high dose (2-5 μg) of αGC in vivo, iNKT cells in the spleen and liver become anergic in terms of the proliferation and cytokine production to subsequent stimulation. In this study, we monitor how iNKT anergy is induced. Anergized iNKT cells dramatically reduced the expression of IL-2Rα, and exogenous IL-2 restored the ability to proliferate and produce IL-4 but not to produce IFN-γ. Anergized iNKT cells expressed high levels of programmed death-1 (PD-1). However, iNKT cells in PD-1-deficient mice became anergic as a result of αGC injection, as do normal mice. Furthermore, anti-PD-1 blocking mAb was unable to restore their responsiveness. When iNKT cells were stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28, they produced cytokines in a dose-dependent manner. Unlike in naive CD4 T cells, the strong TCR-mediated signaling with co-stimulation renders them anergic to any subsequent stimulation with αGC and spleen dendritic cells (DCs). Moreover, iNKT cells also became anergic after stimulation with phorbol-12-myristate-13-acetate + ionophore. Finally, the injection of αGC-pulsed DCs was more potent in inducing anergy than B cells. These results indicate that strong TCR-mediated activation with co-stimulation provides signals that induce the anergic state in iNKT cells.

The C-type lectin SIGNR3 is a mouse homologue of human DC-SIGN, which shares carbohydrate-binding specificity with human DC-SIGN. However, the expression profile of SIGNR3 is largely unknown. To examine the expression of SIGNR3 in immune cells, we generated SIGNR3-specific mAb and investigated SIGNR3 expression in vivo. SIGNR3 was expressed on a fraction of MHC II(+) DCs and Mϕs in the dermis and CD115(+)Ly6C(int-low) monocytes in the blood and BM. In the LNs, SIGNR3(+) cells localized adjacent to PNAd(+) HEV-like vessels. They were also found in interfollicular regions in sLNs but not mLNs. Those SIGNR3(+) cells expressed CD11b and variable levels of CD11c and MHC II. As in LNs, SIGNR3 was expressed on a large proportion of the CD11b(+)CD11c(int-high) cells in the spleen. In the lung, SIGNR3(+) cells belonged to the CD11b(+)CD11c(int) population, and Mϕs in the airway and lung faintly expressed SIGNR3. When PKH67-labeled CD115(+)Ly6C(high) BM monocytes were transferred into normal recipients, they up-regulated SIGNR3 expression along with the decrease in Ly6C expression during the circulation and upon arrival at the peripheral LNs through HEV. In addition, CD11b(high)Ly6C(high) monocytes that entered sLNs differentiated into CD11b(+) DCs in a couple of days, whereas those in the spleen, mLNs, and lung differentiated into CD11c(int) monocytic cells. These results suggest that SIGNR3 is a new differentiation marker for myeloid mononuclear cells and indicate that some DCs, especially in the sLNs, are possibly replenished by Ly6C(high) monocytes.

Interferon regulatory factor (IRF)-2 is a transcription factor involved in type I (IFN- α/β) signaling. It has been reported that IRF-2 deficiency results in various immune dysfunctions. However, the role of IRF-2 in B-cell functions needs to be elucidated. Unlike wild-type (WT) B cells, IRF-2(-/-) B2 cells were refractory to anti-IgM, but not LPS. Such a defect in proliferation was dependent on IFN- α/β receptor (IFNAR). Marginal zone B cells increased in the proportion relative to B2 cells in IRF-2(-/-) mice produced IgM normally to LPS stimulation. However, IRF-2(-/-) B2 cells were defective in IgM production in an IFNAR-independent manner, although both B-cell subsets differentiated phenotypically to plasma cells at elevated efficiencies. Class switch recombination of IRF-2(-/-) B2 cells by LPS plus IL-4 was also impaired. Their reduced IgM production was conceivably due to an inefficient up-regulation of Blimp-1. Consistent with these in vitro observations, specific antibody production in vivo to a T-dependent antigen by B2 cells was severely impaired in IRF-2(-/- )mice. However, a low, but significant, level of IgG was detected at a late time point, and this IgG exhibited comparable binding affinity to that in WT mice. Follicular helper T-cell development and germinal center formation were normal. A similar tendency was observed when µ chain(-/-) mice were reconstituted with IRF-2(-/- )B cells. These results revealed a multi-faceted role of IRF-2 in the function of B cells, particularly B2 cells, through regulating proliferation in an IFNAR-dependent manner and antibody production via up-regulation of Blimp-1.