Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist.

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA.

Engagement of glycosylphosphatidylinositol-anchored proteins results in enhanced mouse and human invariant natural killer T cell responses.

Invariant natural killer T (iNKT) cells are a small subset of lymphocytes that recognize glycolipid antigens in the context of CD1d and consequently produce large quantities of pro-inflammatory and/or anti-inflammatory cytokines. Several transmembrane glycoproteins have been implicated in the co-stimulation of iNKT cell responses. However, whether glycosylphosphatidylinositol (GPI)-anchored proteins can function in this capacity is not known. Here, we demonstrate that antibody-mediated cross-linking of the prototype mouse GPI-anchored protein Thy-1 (CD90) on the surface of a double-negative (CD4⁻CD8⁻) iNKT cell line leads to cytokine production at both the mRNA and protein levels. In addition, Thy-1 triggering enhanced cytokine secretion by iNKT cells that were concomitantly stimulated with α-galactosylceramide (αGC), consistent with a co-stimulatory role for Thy-1 in iNKT cell activation. This was also evident when a CD4+ mouse iNKT cell line or primary hepatic NKT cells were stimulated with αGC and/or anti-Thy-1 antibody. Cross-linking Ly-6A/E, another GPI-anchored protein, could also boost cytokine secretion by αGC-stimulated iNKT cells, suggesting that the observed effects reflect a general property of GPI-anchored proteins. To extend these results from mouse to human cells, we focused on CD55, a GPI-anchored protein that, unlike Thy-1, is expressed on human iNKT cells. Cross-linking CD55 augmented αGC-induced iNKT cell responses as judged by more vigorous proliferation and higher CD69 expression. Collectively, these findings demonstrate for the first time that GPI-anchored proteins are able to co-stimulate CD1d-restricted, glycolipid-reactive iNKT cells in both mice and humans.

CTLA-4Ig blocks the development and progression of citrullinated fibrinogen-induced arthritis in DR4-transgenic mice.

OBJECTIVE: To assess the role of T cells in the mouse model of citrullinated human fibrinogen-induced rheumatoid arthritis (RA) using CTLA-4Ig, an agent that blocks T cell costimulation, which is required for T cell activation. METHODS: Humanized HLA-DRß1*0401-transgenic (DR4-Tg) mice were immunized with Cit-human fibrinogen to induce arthritis. Prior to, and at the onset or peak of, arthritis, the DR4-Tg mice were treated with CTLA-4Ig or control human IgG1 or were left untreated. Arthritis development and progression were monitored by measuring ankle swelling with calipers and by assessing histopathologic changes. The immune responses to the citrullinated antigens and the corresponding unmodified antigens, as well as the arthritogenicity of lymphocytes from these mice, were examined. The latter was performed using lymphocyte transfers from CTLA-4Ig-treated or control mice via intraperitoneal injection into naive DR4-Tg mice. Recipient mice also received an intraarticular injection of Cit-human fibrinogen, unmodified human fibrinogen, or vehicle. RESULTS: CTLA-4Ig-treated, but not human IgG1-treated, arthritic mice had significantly reduced ankle swelling and pathologic joint damage. Treatment with CTLA-4Ig, but not human IgG1, suppressed Cit-human fibrinogen-induced T cell activation, including citrulline-specific T cell activation, when given prior to disease onset. Transfer of splenic lymphocytes from untreated or human IgG1-treated arthritic mice caused arthritis in recipients, and this occurred when Cit-human fibrinogen, but not unmodified fibrinogen, was deposited into the joint. Splenocytes from CTLA-4Ig-treated mice were unable to transfer arthritis. CONCLUSION: Activated citrulline-specific T cells play a direct role in the development and progression of arthritis in this model of Cit-human fibrinogen-induced RA.

Altered immunodominance hierarchies of influenza A virus-specific H-2(b)-restricted CD8+ T cells in the absence of terminal deoxynucleotidyl transferase.

Immunodominance is considered an obstacle to successful T cell-based vaccination, and constant efforts are made to uncover the underlying mechanisms for this phenomenon. We have examined the contribution of terminal deoxynucleotidyl transferase (TdT), whose function accounts for approximately 90% of T cell receptor diversity, to dominance hierarchies of H-2(b)-restricted flu-specific T(CD8+). Using intracellular cytokine staining to quantitatively detect epitope-specific T(CD8+), we demonstrate that TdT-deficient mice exhibit a distinct hierarchical pattern in their primary and recall T(CD8+) responses to influenza A viruses, which results from skewed responsiveness towards select influenza epitopes. Our data establish a link between TdT and immunodominance in H-2(b)-restricted antiviral T(CD8+) responses.

Prevalence of hypoglycemia during oral glucose tolerance testing in adults with cystic fibrosis and risk of developing cystic fibrosis-related diabetes.

BACKGROUND: Hypoglycemia in cystic fibrosis (CF) patients during the oral glucose tolerance test (OGTT) has been reported; however, these patients have not been well-characterized. Few studies have examined whether hypoglycemia during the OGTT increases the risk of developing CF-related diabetes (CFRD). Objectives of this study were to describe the characteristics of CF patients with hypoglycemia during the OGTT and to determine the incidence and time to development of CFRD in those with hypoglycemia. METHODS: This cohort study included 466 adults with CF at the Toronto Adult CF Clinic between 1996 and 2015. Subjects were classified into two groups based on their plasma glucose (PG) level 2 h after a 75 g OGTT: hypoglycemia (PG ≤ 3.9 mmol/L) or no hypoglycemia (PG > 3.9 mmol/L). Clinical and demographic data were collected from the clinic visit closest to the OGTT. Differences between groups were assessed using Fisher's exact test or Mann-Whitney-Wilcoxon test. RESULTS: 138 patients (29.6%) experienced hypoglycemia during the OGTT. More males experienced hypoglycemia compared to no hypoglycemia (69.6% vs. 54.6% respectively; p = 0.003). Those who were heterozygous deltaF508 were more likely to experience hypoglycemia (p = 0.006). Subjects who experienced hypoglycemia were less likely to develop CFRD at ten years compared to no hypoglycemia (12.0% vs. 42.1%, respectively; p < 0.001). CONCLUSIONS: Hypoglycemia following OGTT is common in CF however the 10 year risk of developing CFRD in these patients was low. Males and those who were heterozygous deltaF508 were at higher risk for hypoglycemia.

Longitudinal trends in nutritional status and the relation between lung function and BMI in cystic fibrosis: a population-based cohort study.

BACKGROUND: A high-calorie diet has been a standard of care in cystic fibrosis (CF) for >3 decades. However, energy requirements may have changed with new treatments and milder genotypes. OBJECTIVES: The objectives of this study were to describe longitudinal trends in nutritional status and to evaluate the relation between nutritional status and lung function. DESIGN: This longitudinal cohort study included 909 individuals followed at the Adult CF Clinic in Toronto from 1985 to 2011. Nutritional status was classified on the basis of WHO BMI guidelines. Multivariable linear regression with the use of generalized estimating equations was applied to evaluate the relation between BMI and lung function. RESULTS: The proportion of underweight individuals decreased from 20.6% before 1990 to 11.1% in the most recent decade, whereas the proportion of overweight and obese subjects increased from 7.0% to 18.4% (P < 0.001). Overweight and obese subjects were older, had better lung function, had milder genotypes, and were more often male and pancreatic sufficient. Multivariable regression analyses showed that within the underweight group, an increase in BMI resulted in improved lung function, whereas this effect was half of that in overweight individuals. The greatest advantage of improved nutrition on lung function was observed in the underweight group and in pancreatic- insufficient patients. CONCLUSIONS: Modification to a high-fat diet may be required in some individuals with CF to optimize nutritional health. Higher BMI is associated with improvements in lung function, although the lung function benefit of increasing one's BMI (in kg/m(2)) to >25 is small and needs to be balanced against the known health risks of obesity.