IL-10 produced by induced regulatory T cells (iTregs) controls colitis and pathogenic ex-iTregs during immunotherapy.

"Natural" regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. "Induced" regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1(-/-) mice by the adoptive transfer of naive CD4(+) T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ∼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy.

[Construction of a siRNA plasmid for knockdown of coronin-1].

AIM: To construct a siRNA plasmid to knockdown Coronin-1. METHODS: The cDNA of coronin-1 was amplified by RT-PCR from the total RNA of macrophage, and then inserted into pSEB-HUS vector to generate pSEB-HUS-C plasmid. Three synthesized siRNAs targeting Coronin-1 were cloned into pSEB-HUS-C respectively, resulting in the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids. These plasmids were transiently transfected into A549, and the Coronin-1 level was detected by RT-PCR, Real time PCR and Western blot. RESULTS: All plasmids were successfully constructed as confirmed by restriction enzyme digestion and DNA sequencing. The pSEB-HUS-C3 vector had the most significant knockdown effect on Coronin-1, with 75.9% inhibition at mRNA level and 75.1% inhibition at protein level. CONCLUSION: A siRNA plasmid targeting Coronin-1 was successfully constructed and validated for its knockdown effect, which will serve as a loss-of-function tool for the further mechanistic study of Coronin-1 in tuberculosis pathology.

Methods to identify and characterize different NK cell receptors and their ligands.

Different cellular immune responses are modulated by the cross talk between activating and inhibitory signaling pathways initiated via different cell surface receptors. Similarly, the killing of NK cells is controlled by multiple activating and inhibitory surface receptors. In humans, the major NK triggering receptors, identified so far, include NKp80, 2B4 NKG2D, and CD16 and the natural cytotoxic receptors (collectively named NCRs) include NKp46, NKp44, and NKp30. The two major families of MHC-specific inhibitory receptors identified in humans are the Ig superfamily (KIR and LIR) and the C-type lectin (CD94/NKG2A) receptor superfamily. The different inhibitory receptors show diverse specificity and discriminate between different class I MHC proteins. Much is known about the function and expression patterns of the different NK cell receptors, but the ligand identity of several of the activating NK cell receptors is yet to be discovered. This chapter introduces several research tools that can be used to uncover the identities of different ligands for NK cell receptors.

Anchorage of vinculin to lipid membranes influences cell mechanical properties.

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944-978) and the unstructured C-terminal arm (residues 1052-1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(-/-) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinDeltaC, residues 1-1051). MEF-vinDeltaC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinDeltaC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinDeltaC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.

A novel mutation in IFN-gamma receptor 2 with dominant negative activity: biological consequences of homozygous and heterozygous states.

We identified two siblings homozygous for a single base pair deletion in the IFN-gammaR2 transmembrane domain (791delG) who presented with multifocal Mycobacterium abscessus osteomyelitis (patient 1) and disseminated CMV and Mycobacterium avium complex infection (patient 2), respectively. Although the patients showed no IFN-gammaR activity, their healthy heterozygous parents showed only partial IFN-gammaR activity. An HLA-identical bone marrow transplant from the mother led patient 1 to complete hemopoietic reconstitution, but only partial IFN-gammaR function. We cloned and expressed fluorescent fusion proteins of the wild-type IFN-gammaR2, an IFN-gammaR2 mutant previously described to produce a complete autosomal recessive deficiency (278del2), and of 791delG to determine whether the intermediate phenotype in the 791delG heterozygous state was caused by haploinsufficiency or a dominant negative effect. When cotransfected together with the wild-type vector into IFN-gammaR2-deficient fibroblasts, the fusion protein with 791delG inhibited IFN-gammaR function by 48.7 +/- 5%, whereas fusion proteins with 278del2 had no inhibitory effect. Confocal microscopy of 791delG fusion proteins showed aberrant diffuse intracellular accumulation without plasma membrane localization. The fusion protein created by 791delG did not complete Golgi processing, and was neither expressed on the plasma membrane, nor shed extracellularly. The mutant construct 791delG exerts dominant negative effects on IFN-gamma signaling without cell surface display, suggesting that it is acting on pathways other than those involved in cell surface recognition of ligand.

Quantitative peptidomics of pituitary glands from mice deficient in copper transport.

We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.

RyR1/RyR3 chimeras reveal that multiple domains of RyR1 are involved in skeletal-type E-C coupling.

Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the alpha(1S)-DHPR. Most available evidence suggests that the cytoplasmic II-III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the alpha(1S)-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RyR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RyR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RyR1 (chimera Ch-10 aa 1681-2641 and Ch-9 aa 2642-3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RyR1 residues were 1924-2446 (Ch-21) and 2644-3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RyR1 combine to functionally interact with the DHPR during E-C coupling.