Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-γ contributes to modulate immune responses.

CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8(+) T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.

Genotype, phenotype, and outcomes of nine patients with T-B+NK+ SCID.

There are few reports of clinical presentation, genotype, and HCT outcomes for patients with T-B+NK+ SCID. Between 1981 and 2007, eight of 84 patients with SCID who received and/or were followed after HCT at UCSF had the T-B+NK+ phenotype. One additional patient with T-B+NK+ SCID was identified as the sibling of a patient treated at UCSF. Chart reviews were performed. Molecular analyses of IL7R, IL2RG, JAK3, and the genes encoding the CD3 T-cell receptor components δ (CD3D), ε (CD3E), and ζ (CD3Z) were carried out. IL7R mutations were documented in four patients and CD3D mutations in two others. Three patients had no defects found. Only two of nine patients had an HLA-matched related HCT donor. Both survived, and neither developed GVHD. Five of seven recipients of haploidentical grafts survived. Although the majority of reported cases of T-B+NK+ SCID are caused by defects in IL7R, CD3 complex defects were also found in this series and should be considered when evaluating patients with T-B+NK+ SCID. Additional genes, mutations in which account for T-B+NK+ SCID, remain to be found. Better approaches to early diagnosis and HCT treatment are needed for patients lacking an HLA-matched related donor.

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4.

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11(+) recycling and Rab27(+) late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte-target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.

Protein tyrosine kinase and p38 MAP kinase pathways are involved in stimulation of matrix metalloproteinase-9 by TNF-alpha in human monocytes.

Matrix metalloproteinase-9 (MMP-9), through its catalytic and non-catalytic activities, plays critical roles in inflammation, tumor invasion and angiogenesis. Human monocytes actively involved in inflammatory and tumoral states secrete proMMP-9 (92kDa). Endogenous TNF-alpha stimulates MMP-9 gene transcription in monocytes through NF-kappaB activation. In this study, we investigated the intracellular signaling pathways underlying TNF-alpha/NF-kappaB-dependent expression of MMP-9 in monocytes using chemical inhibitors that specifically inhibit distinct kinase pathways. We confirmed the expression of MMP-9 by reverse transcription chain reaction (RT-PCR), ELISA and gelatin zymography. PGE2/cAMP inhibitor indomethacin, PI-3K inhibitor wortmannin, PKC inhibitor bisindolylmaleimide and PKA inhibitor H-89 did not affect the levels of released MMP-9. In contrast, MMP-9 mRNA and protein expression was down-regulated by p38 MAPK inhibitor SB203580 and protein tyrosine kinase (PTK) inhibitor tyrphostin 25. These inhibitors increased IkappaB-alpha levels, which correlate with decreased NF-kappaB activation. Although SB203580 induced a decrease in TNF-alpha release, addition of exogenous TNF-alpha did not reverse the inhibitory effect of SB203580 toward MMP-9 thus suggesting that SB203580 could modulate down-stream effects of TNF-alpha. In parallel, TIMP-1 levels decreased in the presence of SB203580. Both kinase inhibitors did not influence the maturation pathway of monocytes. Our results indicate that these two inhibitors of p38 MAPK and PTK pathways could be used as combined targets for inhibiting MMP-9 expression in inflamed tissues.

Inhibition of matrix metalloproteinase-9 by interferons and TGF-beta1 through distinct signalings accounts for reduced monocyte invasiveness.

Cytokines may provide signals for regulating human monocyte matrix metalloproteinase-9 (MMP-9) activity. In this study, we investigated the roles of interferons (IFN) type I/II and transforming growth factor-beta1 (TGF-beta1) in MMP-9-mediated invasiveness. MMP-9 antibody and inhibitor, IFNs and TGF-beta1 inhibited monocyte transmigration through Matrigel. IFNs and TGF-beta1 downregulated MMP-9 mRNA, protein and activity levels. The inhibitory action of IFNs was associated with the STAT1/IRF-1 pathway since the JAK inhibitor AG490 blocked STAT1 phosphorylation, IRF-1 synthesis and counteracted the blockade of MMP-9 release. TGF-beta1-mediated MMP-9 inhibition appeared STAT1/IRF-1-independent but reversed by the protein tyrosine kinase inhibitor tyrphostin 25. Our data point out the importance of IFNs and TGF-beta1 in the control of monocyte MMP-9-mediated extravasation.