No abstract

1α,25-Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL-10-secreting CD4 + T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3 + regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3 + and IL-10 + CD4 + T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL-10 at more moderate levels, with little coexpression of these molecules. The Foxp3 + and IL-10 + T-cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL-10. 1α25VitD3 enables the selective expansion of Foxp3 + Treg cells over their Foxp3 − T-cell counterparts. Equally, 1α25VitD3 maintains Foxp3 + expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4 + Foxp3 + T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL-10 + and Foxp3 + Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.Keywords: 1α,25-Dihydroxyvitamin D3 r Asthma r Immune regulation r Regulatory T cells Supporting Information available online IntroductionConsiderable interest exists in the therapeutic potential of regulatory T (Treg) cells to treat a range of immune-mediated patholoCorrespondence: Dr. Catherine M. Hawrylowicz e-mail: catherine.hawrylowicz@kcl.ac.uk gies in humans. This is partly based on evidence obtained from animal models of human disease demonstrating the capacity of Treg cells to control transplant rejection, and to successfully treat autoimmune and allergic disease [1]. Two broad therapeutic * These authors contributed equally to this work. [11]. These studies demonstrate a correlation between therapeutic efficacy and increased frequency or quantities of CD4 + CD25 + T cells, IL-10, TGF-β, and CTLA-4.Our earlier studies have highlighted the capacity of 1α25VitD3 to promote human CD4 + IL-10 secreting Treg cells (IL-10-Treg) in culture both alone [12] and in concert with glucocorticoids such as dexamethasone [13,14]. Furthermore, treatment of severe steroid refractory asthma patients with 1α25VitD3 in vivo directly increased IL-10 gene expression in CD3 + CD4 + T cells [12], and restored the impaired steroid-induced IL-10 response in CD4 + cells in vitro [14,15].The present study was designed to further investigate the mechanisms underlying the therapeutic potential of 1α25VitD3 in the context of asthmatic disease, and to determine effects on the induction of both IL-10 + and Foxp3 + T cells. Specifically, we have examined the effects of 1α25VitD3 on total, unfractionated CD4 + T-cell populations, r...

No abstract

Changes in monocytes and their subsets (CD14hi/CD16neg, CD14hi/CD16pos and CD14lo/CD16pos) have been described in several diseases. The combination of CD14, CD16 and HLA‐DR has been suggested to discriminate monocytes from the CD16pos/HLA‐DRneg NK‐cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA‐DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66bpos neutrophils, CD56pos NKcells, CD19pos B‐cells, and CD3pos T‐cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA‐DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA‐DR expression]. Bland‐Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14hi/CD16pos monocytes was higher (7% vs 4%) whilst the percentage of CD14lo/CD16pos was lower (1.9% vs. 7%) (P ≤ 0.001); HLA‐DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA‐DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody‐fluorochrome “backbone” targeting HLA‐DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody‐fluorochrome “backbone” of HLA‐DR, CD14, and CD16. CD16pos monocytes continue to constitutively express HLA‐DR even in conditions where HLA‐DR is pathologically reduced on CD14hi/CD16neg monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico‐pathological diseases may allow the development of novel biomarkers or therapeutic strategies. © 2012 International Society for Advancement of Cytometry

The immune system contains natural regulatory T cells that control the magnitude of the immune response during physiologic and pathologic conditions. Although this suppressive function was historically attributed to CD8 T cells, most recent reports have focused on natural regulatory CD4 T cells. In the present study, we describe a new subset of natural CD8 regulatory T cells in normal healthy animals. This subset expresses low levels of CD45RC at its surface (CD45RC low ); produces mainly interleukin-4 (IL-4), IL-10, and IL-13 cytokines upon in vitro stimulation; expresses Foxp3 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4); and is not cytotoxic against allogeneic targets. This subset suppresses the proliferation and differentiation of autologous CD4 T cells into type-1 cytokines producing T cells after stimulation with allogeneic accessory cells. We also provide evidence that this regulatory subset mediates its suppression by cell-to-cell contact and not through secretion of suppressive cytokines. Finally, the regulatory activity of CD8 CD45RC low cells is also demonstrated in vivo in a rat model of CD4-dependent graft-versus-host disease. Collectively, these data demonstrate for the first time that freshly isolated rat CD8 CD45RC low T cells contain T cells with regulatory properties, a result that enlarges the general picture of T-cell-mediated regulation. ( IntroductionThe delicate balance between pathogen-induced effector immunologic functions and natural self-tolerance mechanisms is of vital importance for preserving the integrity of a host in the course of an immune response. In different species of rodents and in humans, there is compelling evidence that the regulation of the magnitude of protective immunity to foreign antigens as well as the control of autoaggressive immune reactions are ensured by regulatory CD4 and CD8 T lymphocytes that display anti-inflammatory and antiproliferative functions. Convergent evidence indicates that multiple subtypes of regulatory T cells exist and that their regulatory activities are mediated either by immunosuppressive cytokines or by contact-dependent mechanisms. [1][2][3][4][5][6][7][8] CD45 is a transmembrane tyrosine phosphatase expressed as isoforms of different molecular weight, which result in the differential splicing of 3 exons (A, B, and C) encoding part of the N-terminal extracellular domain. In the rat, CD45RC expression levels define 2 subpopulations of CD4 T cells with different cytokine profiles and functions. 3,[9][10][11][12] Functional analyses of CD45RC high and CD45RC low CD4 T cells have demonstrated that important regulatory interactions occur between these subsets in vivo. 11,13,14 For example, the adoptive transfer of CD45RC high CD4 T cells from congenic euthymic donors to nude rats induces a fatal wasting disease, while the transfer of both subpopulations, or of the CD45RC low cells alone, has no effect. 11 Similar results were obtained using mouse CD4 T cells fractionated on the basis of CD45RB expression. 15 It has also been demo...

Liver transplantation (LT) is a successful treatment for patients with liver failure. However, organ shortage results in over 11% of patients losing their chance of a transplant attributed to liver decompensation (LD) and death. Ischemia/reperfusion injury (IRI) following conventional cold storage (CS) is a major cause of injury leading to graft loss after LT. Normothermic machine perfusion (NMP), a method of organ preservation, provides oxygen and nutrition during preservation and allows aerobic metabolism. NMP has recently been shown to enable improved organ utilization and posttransplant outcomes following a phase I and a phase III randomized trial. The aim of the present study is to assess the impact of NMP on reducing IRI and to define the underlying mechanisms. We transplanted and compared 12 NMP with 27 CS‐preserved livers by performing gene microarray, immunoprofiling of hepatic lymphocytes, and immunochemistry staining of liver tissues for assessing necrosis, platelet deposition, and neutrophil infiltration, and the status of steatosis after NMP or CS prereperfusion and postreperfusion. Recipients receiving NMP grafts showed significantly lower peak aspartate aminotransferase (AST) levels than those receiving CS grafts. NMP altered gene‐expression profiles of liver tissue from proinflammation to prohealing and regeneration. NMP also reduced the number of interferon gamma (IFN‐γ) and interleukin (IL)‐17–producing T cells and enlarged the CD4posCD25highCD127negFOXP3pos regulatory T cell (Treg) pool. NMP liver tissues showed less necrosis and apoptosis in the parenchyma and fewer neutrophil infiltration compared to CS liver tissues. Conclusion: Reduced IRI in NMP recipients was the consequence of the combination of inhibiting inflammation and promoting graft regeneration.

The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune-mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases while BN rats are highly susceptible to Th2-mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC-dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC-independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL-4, IL-13, IL-10 and TGF-beta in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1-mediated experimental autoimmune encephalomyelitis, and to Th2-mediated disorders triggered by gold salts as well as the difference in the CD4SRChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

BackgroundAssociations between vitamin D status and childhood asthma are increasingly reported, but direct causation and mechanisms underlying an effect remain unknown. We investigated the effect of early-life vitamin D deficiency on the development of murine neonatal allergic airways disease (AAD).MethodsIn utero and early-life vitamin D deficiency was achieved using a vitamin D-deficient diet for female mice during the third trimester of pregnancy and lactation. Offspring were weaned onto a vitamin D-deficient or vitamin D-replete diet, and exposure to intranasal house dust mite (HDM) or saline was commenced from day 3 of life for up to 6 weeks, when airway hyper-responsiveness (AHR), airway inflammation and remodelling were assessed.ResultsNeonatal mice that had in utero and early-life vitamin D deficiency had significantly increased pulmonary CD3+CD4+T1ST2+ cells and reduced CD4+IL-10+ cells. This effect was enhanced following HDM exposure. AHR in HDM-exposed mice was unaffected by vitamin D status. Introduction of vitamin D into the diet at weaning resulted in a significant reduction in serum IgE levels, reduced pulmonary eosinophilia and peri-bronchiolar collagen deposition.ConclusionPeri-natal vitamin D deficiency alone has immunomodulatory effects including Th2 skewing and reduced IL-10-secreting T regulatory cells, exaggerated with additional allergen exposure. Vitamin D deficiency in early life does not affect AHR, but contributes to disease severity with worse eosinophilic inflammation and airway remodelling. Importantly, supplementation with vitamin D improves both of these pathological abnormalities.