Youn-Soo Hahn

Different Potentials of γδ T Cell Subsets in Regulating Airway Responsiveness: Vγ1+ Cells, but Not Vγ4+ Cells, Promote Airway Hyperreactivity, Th2 Cytokines, and Airway Inflammation

Allergic airway inflammation and hyperreactivity are modulated by γδ T cells, but different experimental parameters can influence the effects observed. For example, in sensitized C57BL/6 and BALB/c mice, transient depletion of all TCR-δ+ cells just before airway challenge resulted in airway hyperresponsiveness (AHR), but caused hyporesponsiveness when initiated before i.p. sensitization. Vγ4+ γδ T cells strongly suppressed AHR; their depletion relieved suppression when initiated before challenge, but not before sensitization, and they suppressed AHR when transferred before challenge into sensitized TCR-Vγ4−/−/6−/− mice. In contrast, Vγ1+ γδ T cells enhanced AHR and airway inflammation. In normal mice (C57BL/6 and BALB/c), enhancement of AHR was abrogated only when these cells were depleted before sensitization, but not before challenge, and with regard to airway inflammation, this effect was limited to C57BL/6 mice. However, Vγ1+ γδ T cells enhanced AHR when transferred before challenge into sensitized B6.TCR-δ−/− mice. In this study Vγ1+ cells also increased levels of Th2 cytokines in the airways and, to a lesser extent, lung eosinophil numbers. Thus, Vγ4+ cells suppress AHR, and Vγ1+ cells enhance AHR and airway inflammation under defined experimental conditions. These findings show how γδ T cells can be both inhibitors and enhancers of AHR and airway inflammation, and they provide further support for the hypothesis that TCR expression and function cosegregate in γδ T cells.

Distribution and leukocyte contacts of γδ T cells in the lung

Pulmonary γδ T cells protect the lung and its functions, but little is known about their distribution in this organ and their relationship to other pulmonary cells. We now show that γδ and αβ T cells are distributed differently in the normal mouse lung. The γδ T cells have a bias for nonalveolar locations, with the exception of the airway mucosa. Subsets of γδ T cells exhibit further variation in their tissue localization. γδ and αβ T cells frequently contact other leukocytes, but they favor different cell‐types. The γδ T cells show an intrinsic preference for F4/80+ and major histocompatibility complex class II+ leukocytes. Leukocytes expressing these markers include macrophages and dendritic cells, known to function as sentinels of airways and lung tissues. The continuous interaction of γδ T cells with these sentinels likely is related to their protective role.

MHC class I-dependent Vγ4+ pulmonary T cells regulate αβ T cell-independent airway responsiveness

Mice exposed to aerosolized ovalbumin (OVA) develop increased airway responsiveness when deficient in γδ T cells. This finding suggests that γδ T cells function as negative regulators. The regulatory influence of γδ T cells is evident after OVA-sensitization and -challenge, and after OVA-challenge alone, but not in untreated mice. With aerosolized Abs to target pulmonary T cells, we now demonstrate that negative regulation of airway responsiveness is mediated by a small subpopulation of pulmonary γδ T cells. These cells express Vγ4 and depend in their function on the presence of IFN-γ and MHC class I. Moreover, their effect can be demonstrated in the absence of αβ T cells. This novel type of negative regulation seems to precede the development of the adaptive, antigen-specific allergic response.

Vγ4+ γδ T Cells Regulate Airway Hyperreactivity to Methacholine in Ovalbumin-Sensitized and Challenged Mice

The Vγ4+ pulmonary subset of γδ T cells regulates innate airway responsiveness in the absence of αβ T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vγ4+ cells preferentially increased in number following airway challenge. Depletion of Vγ4+ cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vγ1+ cells had no effect on AHR, and depletion of all TCR-δ+ cells was no more effective than depletion of Vγ4+ cells alone. Adoptively transferred pulmonary lymphocytes containing Vγ4+ cells inhibited AHR, but lost this ability when Vγ4+ cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vγ4+ cells, although cytokine-producing αβ T cells in the lung increased. These findings establish Vγ4+ γδ T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.

A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice

ABSTRACT The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N15-X16-T17, which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substiting for N15 and/or T17 and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an ≈20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

Subset‐specific, uniform activation among Vγ6/Vδ1+ γδ T cells elicited by inflammation

The Vγ6/Vδ1+ cells, the second murine γδ T cell subset to arise in the thymus, express a nearly invariant T cell receptor (TCR), colonize select tissues, and expand preferentially in other tissues during inflammation. These cells are thought to help in regulating the inflammatory response. Until now, Vγ6/Vδ1+ cells have only been detectable indirectly, by expression of Vγ6‐encoding mRNA. Here, we report that 17D1, a monoclonal antibody, which detects the related epidermis‐associated Vγ5/Vδ1+ TCR, will also bind the Vγ6/Vδ1+ cells if their TCR is first complexed to an anti‐Cδ antibody. Features of this special condition for recognition suggest the possibility that an alternate structure exists for the Vγ6/Vδ1 TCR, which is stabilized upon binding to the anti‐Cδ antibody. Using the 17D1 antibody as means to track this γδ T cell subset by flow cytometry, we discovered that the response of Vγ6/Vδ1+ cells during inflammation often far exceeds that of other subsets and that the responding Vγ6/Vδ1+ cells display a strikingly uniform activation/memory phenotype compared with other γδ T cell subsets.

Effect of Lactobacillus sakei supplementation in children with atopic eczema–dermatitis syndrome

BACKGROUND Probiotics have been suggested to be useful in children with atopic eczema-dermatitis syndrome (AEDS). OBJECTIVE To assess the clinical effect of Lactobacillus sakei supplementation in children with AEDS. METHODS In a double-blind, placebo-controlled trial, children aged 2 to 10 years with AEDS with a minimum SCORing of Atopic Dermatitis (SCORAD) score of 25 were randomized to receive either daily L sakei KCTC 10755BP or daily placebo supplementation for 12 weeks. Changes in SCORAD scores and serum chemokine levels from baseline were evaluated. RESULTS Eighty-eight children were enrolled, and 45 were allocated to probiotic treatment. Seventy-five children completed the study, with 4 dropouts in the probiotic group and 9 in the placebo group. At week 12, SCORAD total scores adjusted by pretreatment values were lower after probiotic treatment than after placebo treatment (P = .01). There was a 31% (13.1-point) improvement in mean disease activity with probiotic use compared with a 13% (5.2-point) improvement with placebo use (P = .008). Significant differences in favor of probiotic treatment were also observed in proportions of patients achieving improvement of at least 30% and 50%. Compared with placebo, probiotic administration was associated with lower pretreatment-adjusted serum levels of CCL17 and CCL27 (P =.03 for both), which were significantly correlated with SCORAD total score (r = 0.59 and 0.63, respectively; P < .001). CONCLUSIONS Supplementation of L sakei in children with AEDS was associated with a substantial clinical improvement and a significant decrease in chemokine levels, reflecting the severity of AEDS.

Analysis of γδ T Cell Functions in the Mouse

Mouse models of disease and injury have been invaluable in investigations of the functional role of γδ T cells. They show that γδ T cells engage in immune responses both early and late, that they can function both polyclonally and as peripherally selected clones, and that they can be effector cells and immune regulators. They also suggest that functional development of γδ T cells occurs stepwise in thymus and periphery, and that it is governed by γδ TCR-signaling and other signals. Finally, they indicate that γδ T cell functions often segregate with TCR-defined subsets, in contrast to conventional T cells. From the functional studies in mice and other animal models, γδ T cells emerge as a distinct lymphocyte population with a unique and broad functional repertoire, and with important roles in Ab responses, inflammation and tissue repair. They also are revealed as a potentially useful target for immune intervention.

Mismatched Antigen Prepares γδ T Cells for Suppression of Airway Hyperresponsiveness

γδ T cells suppress airway hyperresponsiveness (AHR) induced in allergen-challenged mice but it is not clear whether the suppression is allergen specific. The AHR-suppressive cells express TCR-Vγ4. To test whether the suppressive function must be induced, we adoptively transferred purified Vγ4+ cells into γδ T cell-deficient and OVA-sensitized and -challenged recipients (B6.TCR-Vγ4−/−/6−/−) and measured the effect on AHR. Vγ4+ γδ T cells isolated from naive donors were not AHR-suppressive, but Vγ4+ cells from OVA-stimulated donors suppressed AHR. Suppressive Vγ4+ cells could be isolated from lung and spleen. Their induction in the spleen required sensitization and challenge. In the lung, their function was induced by airway challenge alone. Induction of the suppressors was associated with their activation but it did not alter their ability to accumulate in the lung. Vγ4+ γδ T cells preferentially express Vδ4 and -5 but their AHR-suppressive function was not dependent on these Vδs. Donor sensitization and challenge not only with OVA but also with two unrelated allergens (ragweed and BSA) induced Vγ4+ cells capable of suppressing AHR in the OVA-hyperresponsive recipients, but the process of sensitization and challenge alone (adjuvant and saline only) was not sufficient to induce suppressor function, and LPS as a component of the allergen was not essential. We conclude that AHR-suppressive Vγ4+ γδ T cells require induction. They are induced by allergen stimulation, but AHR suppression by these cells does not require their restimulation with the same allergen.

Utility of fractional exhaled nitric oxide (FENO) measurements in diagnosing asthma

BACKGROUND To facilitate the use of fractional exhaled nitric oxide (F(E)NO) as a clinical test, F(E)NO measurements need more clarification. AIM We sought to evaluate the yield of F(E)NO measurement for the diagnosis of asthma and identify the determinants of F(E)NO in children. METHODS Two hundred forty five consecutive steroid-naïve patients aged 8-16 years with symptoms suggestive of asthma were included. Children were evaluated using F(E)NO measurements, questionnaires, skin prick tests, spirometries, and methacholine challenge tests. RESULTS Asthma was diagnosed in 167 children. The sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) of F(E)NO measurements for the diagnosis of asthma at the best cutoff value of 22 ppb were 56.9%, 87.2%, 90.5%, and 48.6%, respectively. At a cutoff value of 42 ppb, specificity and PPV were all 100% but at the cost of very low sensitivity (23.4%) and NPV (37.9%). Both atopy and asthma were identified as independent risk factors associated with high F(E)NO. The association of asthma with high F(E)NO was found only in atopic children because F(E)NO was low in non-atopic children regardless of asthma status. Although highest F(E)NO was observed in atopic asthmatic patients, 28% of these patients had F(E)NO values lower than 22 ppb. CONCLUSION Atopic asthmatic patients with low F(E)NO values and non-atopic asthmatic patients were responsible for false-negative cases that might contribute to low sensitivity of F(E)NO measurements in diagnosing asthma. High specificity of F(E)NO measurements may help identify patients with atopic asthma among subjects with respiratory symptoms.