Comparison of blood and synovial fluid th17 and novel peptidase inhibitor 16 Treg cell subsets in juvenile idiopathic arthritis.

OBJECTIVE: Early recognition and treatment of juvenile idiopathic arthritis (JIA) can prevent joint damage and minimize side effects of medication. The balance between proinflammatory and antiinflammatory mechanisms is known to be important in JIA, and we therefore investigated T cell subsets including Th cells, autoaggressive Th17 cells, and regulatory T cells (Treg), including a novel Treg subset in peripheral blood (PB) and synovial fluid (SF) of patients with JIA. METHODS: Fifty children with JIA were enrolled in our study. Frequency, phenotype, and function of T lymphocytes in PB and SF were characterized using flow cytometry. Migration capabilities of PB and SF cells were compared. RESULTS: Synovial T cells showed different phenotype and function compared with PB T cells, with an increased proportion of memory T cells, expression of CCR4, CCR5, CXCR3, interleukin 23R, and an increased ratio of Th17 to Treg. Although Treg were increased in SF compared with the PB, we found a significant decrease in the numbers of peptidase inhibitor 16 (PI16)+ Treg in active joints compared with peripheral blood. Coexpression of CCR4 and CCR6 was reduced on PI16+ Treg in PB and SF of patients with JIA compared with healthy children, however the ability of these cells to migrate toward their ligands was unaffected. CONCLUSION: This is a comprehensive characterization of novel PI16+ Treg and Th17 cells in matched blood and synovial fluid samples of patients with JIA. Despite an increased number of Treg within the inflamed joint, lower numbers of PI16+ Treg but high numbers of Th17 cells might contribute to the inability to control disease.

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20.

The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.

Expression of Toll-like receptors by neonatal leukocytes.

The immune system of neonates is poorly developed; this increases the susceptibility of neonates to infection. For neonates to counter infection effectively, they first need to recognize the presence of pathogens. Toll-like receptors (TLR) are a family of pattern recognition receptors that alert the host to the presence of invading pathogens. To determine whether differences in TLR expression by leukocytes compensate for immunologic immaturity in neonates, TLR expression by monocytes and T lymphocytes from adults and neonates was compared. Expression of TLR1, TLR2, TLR3, TLR4, TLR8 and TLR9 by monocytes and T lymphocytes was detected with antibodies by flow cytometry. TLR1, TLR2, TLR3, TLR4, TLR8 and TLR9 expression by monocytes was detected in adults and neonates. TLR2, TLR3, TLR4, TLR8 and TLR9 expression by T lymphocytes was detected in adults and neonates. Monocytes and T lymphocytes from neonates are capable, like adults, of recognizing the presence of pathogens through TLR.

Expression of toll-like receptors on B lymphocytes.

Toll-like receptors (TLRs) are a family of trans-membrane receptors that play an important role in the innate immune system. Most studies examining the cellular expression of TLRs on immune cells have focussed on neutrophils, monocytes and dendritic cells, but there is little evidence of TLRs being expressed on lymphocytes. Using 3-colour flow cytometry, expression of TLR-1, TLR-2, TLR-3, TLR-4, and TLR-9 on peripheral blood lymphocyte populations was determined. Further examination of TLRs on CD5- and CD5+ CD19+ B cell subsets was performed. The binding of TLR1 and TLR9 antibodies was detected on 15-90% of resting B cells, but not on resting T-cells. The higher expression of TLR1 and TLR9 on CD5+ B cells compared to CD5- B cells may reflect the role of B1 cells in more primitive, less specific antibody responses.

Characterisation of the protein composition of peripheral blood mononuclear cell microsomes by SDS-PAGE and mass spectrometry.

Approximately 340 leucocyte plasma membrane proteins have been characterised by the eight Human Leucocyte Differentiation Antigen workshops held between 1982 and 2004, based primarily on their reactivity with monoclonal antibodies. The human genome is predicted to encode approximately 34,000 cDNA transcripts, of which between 15% and 20% are predicted to contain one or more transmembrane helices. We have used SDS-PAGE separation coupled with mass spectrometry-based peptide mass tag identification to identify novel plasma membrane proteins in microsome preparations prepared from mononuclear cells obtained from human peripheral blood. A total of 361 distinct proteins were identified in a single preparation, including 37 known leucocyte plasma membrane proteins, 27 potential novel plasma membrane proteins whose expression on PBMC is poorly characterised, and 51 other proteins for which the subcellular location could not be determined. Expression analysis using cDNA panels indicates that several of these novel plasma membrane proteins are differentially expressed in lymphocyte subsets. These results show that previously unidentified lymphocyte plasma membrane proteins can be identified using this approach.

New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin.

In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.

Detection of low-abundance membrane markers by immunofluorescence--a comparison of alternative high-sensitivity methods and reagents.

The analysis of membrane molecules using antibodies detected by immunofluorescence staining and flow cytometry is used widely in research and diagnostic immunology. Conventional staining techniques readily detect molecules present at concentrations of around 2000 molecules per cell, but some molecules are expressed and function at much lower abundance. We described previously a method for the detection of molecules present at 100 molecules per cell or less based on the use of phycoerythrin as the fluorophore, a three-layer amplification process, and careful selection of available reagents. In recent years, a number of new reagents, fluorophores and kits, have become available, some of them intended for high-sensitivity applications. In this paper, a number of these reagents have been compared with the published method. While some of the reagents gave variable results or high nonspecific staining in our hands, several reagents were comparable with the published method. Furthermore, the new fluorophores allow improved simultaneous detection of two low-abundance markers.

Tolerization as a tool for generating novel monoclonal antibodies.

Standard hybridoma production involves the fusion of spleen cells from an immunized mouse with a non-secretory murine myeloma cell line. While this technology has provided numerous reagents that are highly valuable, demand is now increasing for monoclonal antibodies which can distinguish between closely related antigens. Induction of tolerance towards common antigens enables the recovery of high-specificity reagents that have previously proved elusive. This review details a number of strategies using either complex protein mixtures or purified proteins as tolerogens and subsequent immunization with a closely related immunogen.