SUMMARY Type I diabetes is a T cell-mediated autoimmune disease, characterized by lymphocytic infiltration of the pancreatic islets. It is currently thought that islet antigen-specificity is not a requirement for islet entry and that diabetogenic T cells can recruit a heterogeneous bystander T cell population. We tested this assumption directly by generating TCR retrogenic mice expressing two different T cell populations. By combining diabetogenic and non-diabetogenic and/or non-autoantigen specific T cells, we demonstrate that bystander T cells cannot accumulate in the pancreatic islets. Autoantigen specific T cells which accumulate in islets, but do not cause diabetes, were also unaffected by the presence of diabetogenic T cells. Additionally, 67% of TCRs cloned from NOD islet-infiltrating CD4+ T cells were able to mediate cell-autonomous islet infiltration and/or diabetes when expressed in retrogenic mice. Therefore islet entry/accumulation appears to be a cell-autonomous and tightly-regulated event and is governed by islet antigen specificity.

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OBJECTIVE—Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing β-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo. RESEARCH DESIGN AND METHODS—We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2β/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared. RESULTS—Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2β/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42–56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice. CONCLUSIONS—Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and β-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

Interleukin (IL)-35 is a newly identified inhibitory cytokine used by T regulatory cells to control T cell–driven immune responses. However, the therapeutic potential of native, biologically active IL-35 has not been fully examined. Expression of the heterodimeric IL-35 cytokine was targeted to β-cells via the rat insulin promoter (RIP) II. Autoimmune diabetes, insulitis, and the infiltrating cellular populations were analyzed. Ectopic expression of IL-35 by pancreatic β-cells led to substantial, long-term protection against autoimmune diabetes, despite limited intraislet IL-35 secretion. Nonobese diabetic RIP-IL35 transgenic mice exhibited decreased islet infiltration with substantial reductions in the number of CD4+ and CD8+ T cells, and frequency of glucose-6-phosphatase catalytic subunit–related protein-specific CD8+ T cells. Although there were limited alterations in cytokine expression, the reduced T-cell numbers observed coincided with diminished T-cell proliferation and G1 arrest, hallmarks of IL-35 biological activity. These data present a proof of principle that IL-35 could be used as a potent inhibitor of autoimmune diabetes and implicate its potential therapeutic utility in the treatment of type 1 diabetes.

We identified a novel phenotype of SF NK cells that is of potential significance in RA. Experiments are now under way to determine the function of these SF NK cells and their potential role in RA.

IL‐19 is a novel, recently identified member of the IL‐10 family of cytokines. We identified IL‐10 as a cytokine that was strongly induced in IL‐19‐stimulated PBMC. IL‐19‐induced IL‐10 secretion was dose‐dependent and could be detected in culture supernatants after 3 h of stimulation. Furthermore, quantitative RT‐PCR analysis demonstrated that IL‐19 stimulation increased the level of IL‐10 mRNA present within cells, suggesting that IL‐19 is a transcriptional activator of IL‐10. IL‐19 was also able to induce its own expression, with IL‐10 potently down‐regulating this IL‐19 ‘auto‐induction’. LPS induction of IL‐19 expression was also regulated by IL‐10, demonstrating that IL‐10 is likely an important regulator of human IL‐19 induction. Maturation of dendritic cells from human PBMC in the presence of IL‐19 resulted in an increase in IL‐10 levels within these cells, whereas IL‐12 was not affected. These results advance our understanding of the function of this novel cytokine and its regulation within the human immune system, in addition to providing a new insight into the control of the important immunoregulatory cytokine, IL‐10.

Lactoferrin is an antimicrobial protein which plays an important role in regulating bacteria that are associated with aggressive periodontitis. Lactoferrin kills directly (via its strongly cationic N-terminal region) and indirectly, through sequestering the iron that bacteria require for growth. As aggressive periodontitis has a strong heritable component, we hypothesized that genetic variation within the lactoferrin gene may play a role in susceptibility to this condition. We have identified and examined a novel, functional, single-point A/G nucleotide mutation causing a threonine/alanine substitution at position 11 (T11A) of the secreted lactoferrin protein. In a pilot case-controlled study of aggressive periodontitis, analysis of 46 African-American patients and 78 controls showed that patients were twice as likely to express the G nucleotide (alanine) allele over controls (60.3 vs 30.4%; P ¼ 0.0007, odds ratio ¼ 2.564, 95% CI ¼ 1. 475-4.459). A Caucasian population of 77 patients and 131 controls showed no such association (P ¼ 0.5201, odds ratio ¼ 0.862, 95% CI ¼ 0.548-1.356). The data presented provide a new insight into the genetic susceptibility to aggressive periodontitis. Genes and Immunity (2005) Lactoferrin is an 80 kDa cationic protein with strong antibacterial and immunomodulatory functions. [1][2][3][4][5] The antibacterial properties were originally attributed solely to its ability to bind the iron necessary for bacterial growth. However, it has been established more recently that lactoferrin also binds to bacteria and kills through direct interactions governed by its strongly basic Nterminal region. [6][7][8][9] In addition to its ability to kill bacteria, lactoferrin can also neutralize endotoxin and inhibit the induction of NFkB in monocytes in response to LPS, resulting in lowered IL-6 and TNF-alpha production. [10][11][12] Lactoferrin is present in high concentrations in saliva and is thought to play a particularly important role in regulation of those bacteria present within the oral cavity. This protein regulates several bacteria that are associated with periodontal disease, including Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Prevotella melaninogenica (Pm) and Actinobacillus actinomycetemcomitans (Aa). [13][14][15][16][17][18] Lactoferrin's effects on Aa, which is considered to have the strongest association with aggressive periodontitis, occurs through direct killing, as well as through inhibition of attachment to epithelial cells and the development of biofilms. 14,19,20 Studies examining the pattern of inheritance of aggressive periodontitis in families suggest a genetic basis for susceptibility. 18,21 The disease is particularly common within African-American populations, being up to 15 times more prevalent than in Caucasians. 21 As lactoferrin has been shown to be active against a number of pathogenic bacteria, including Aa, we hypothesized that single-nucleotide polymorphism (SNPs) within the lactoferrin gene could play a role in the genetic susceptibility that is s...

A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The ؊44 C3G transversion in the DEFB1 gene (which encodes human ␤-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the ؊44 SNP. We used an HP assay to study the distribution of the ؊44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the ؊44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.