Immune memory in CD4+ CD45RA+ T cells.

This study addresses the question of whether human peripheral CD4+ CD45RA+ T cells possess antigen-specific immune memory. CD4+ CD45RA+ T cells were isolated by a combination of positive and negative selection. Putative CD4+ CD45RA+ cells expressed CD45RA (98.9%) and contained < 0.1% CD4+ CD45RO+ and < 0.5% CD4+ CD45RA+ CD45RO+ cells. Putative CD45RO+ cells expressed CD45RO (90%) and contained 9% CD45RA+ CD45RO+ and < 0.1% CD4+ CD45RA+ cells. The responder frequency of Dermatophagoides pteronyssinus-stimulated CD4+ CD45RA+ and CD4+ CD45RO+ T cells was determined in two atopic donors and found to be 1:11,314 and 1:8031 for CD4+ CD45RA+ and 1:1463 and 1:1408 for CD4+ CD45RO+ T cells. The responder frequencies of CD4+ CD45RA+ and CD4+ CD45RO+ T cells from two non-atopic, but exposed, donors were 1:78031 and 1:176,903 for CD4+ CD45RA+ and 1:9136 and 1:13,136 for CD4+ CD45RO+ T cells. T cells specific for D. pteronyssinus were cloned at limiting dilution following 10 days of bulk culture with D. pteronyssinus antigen. Sixty-eight clones were obtained from CD4+ CD45RO+ and 24 from CD4+ CD45RA+ T cells. All clones were CD3+ CD4+ CD45RO+ and proliferated in response to D. pteronyssinus antigens. Of 40 clones tested, none responded to Tubercule bacillus purified protein derivative (PPD). No difference was seen in the pattern of interleukin-4 (IL-4) or interferon-gamma (IFN-gamma) producing clones derived from CD4+ CD45RA+ and CD4+ CD45RO+ precursors, although freshly isolated and polyclonally activated CD4+ CD45RA+ T cells produced 20-30-fold lower levels of IL-4 and IFN-gamma than their CD4+ CD45RO+ counterparts. Sixty per cent of the clones used the same pool of V beta genes. These data support the hypothesis that immune memory resides in CD4+ CD45RA+ as well as CD4+ CD45RO+ T cells during the chronic immune response to inhaled antigen.     

Intracellular IL-5 and T-lymphocyte subsets in atopic and nonatopic bronchial asthma

BACKGROUND: Allergen-stimulated IL-5 production by CD4+ T cells is the key issue in atopic asthma. On the other hand, virus-specific CD8+ T cells produce IL-5 and might play an important role in the pathogenesis of nonatopic asthma. OBJECTIVES: We sought to compare the IL-5-producing T-lymphocyte subsets in the peripheral blood of atopic and nonatopic asthmatic subjects, especially the contribution of IL-5-producing CD8(+) T cells in nonatopic asthma. METHODS: Heparinized blood samples were obtained from subjects with atopic asthma (n = 10), subjects with nonatopic asthma (n = 10), and healthy subjects (n = 10) and stimulated with ionomycin and phorbol myristate acetate in the presence of brefeldin A. Two-color flow cytometric analysis with mAbs to cell-surface antigens and intracellular IL-5 was used to detect the IL-5-producing T-cell subsets. RESULTS: A higher percentage of IL-5-producing CD3(+) T cells was detected in subjects with atopic and nonatopic asthma than that seen in the healthy subjects. The percentage of IL-5-producing CD4(+) T cells was significantly higher in subjects with atopic asthma than in the healthy subjects. The percentage of IL-5-producing CD8(+) T cells was significantly higher in subjects with nonatopic asthma than in the healthy subjects. The percentage of IL-5-producing CD8(+) T cells was higher in subjects with nonatopic asthma than in those with atopic asthma, but the difference was not statistically significant. CONCLUSIONS: CD4(+) T cells are the major source of IL-5 among CD3(+) lymphocytes in subjects with atopic asthma. On the other hand, increased IL-5 production by CD8(+) T cells, as well as by CD4(+) T cells, is a characteristic feature of nonatopic asthma.     

Allergen-induced CD30 expression on T cells of atopic asthmatics

Background The importance of TH₂-type T cell cytokines in atopic disease is widely accepted. CD30, a member of the TNF/NGF receptor superfamily, is expressed on a proportion of activated CD45RO⁺ T cells and has been proposed as a marker for TH₂ phenotype. CD30 ligand-CD30 interaction has been shown to positively influence development of the TH₂ phenotype, and serum levels of soluble CD30 (sCD30) have been used as prognostic markers in HIV, SLE, Epstein-Barr Virus infection and Hodgkin's Lymphoma but not as yet in allergic disease. Objectives To establish if serum levels of sCD30 are elevated in atopic asthma and determine whether allergen-induced proliferation/activation of PBMCs from atopic asthmatics promotes CD30 expression on CD4⁺ T lymphocytes. Further, to determine if expression of CD30 and sCD30 correlate with disease severity. Methods Eighteen atopic asthmatics were each assigned a symptomatic disease score based on symptoms and bronchodilator rescue usage. Serum sCD30 was measured in peripheral blood by ELISA. PBMCs from atopic asthmatics were analysed with flow cytometry to obtain the proportions of CD⁴ T cells expressing CD45RO and CD30. The cells were then cultured for 10 days with IL-2 with or without house dust mite antigen. A proliferation index was recorded and expression of CD30 and CD45RO retested. As a control, stimulation with PHA was used. Results with patients’ PBMCs were compared with results of a parallel analysis of PBMCs from non-atopic healthy controls. Results Serum sCD30 was elevated in the 18 atopic asthmatics compared with a group of normal subjects but levels did not correlate with symptomatic disease activity. CD4CD45RO expression was low (14%) in atopic asthmatic peripheral blood but increased to 41% after 10 days culture with allergen. The CD4:CD8 ratio increased after Der p stimulation. A significant rise in the percentage of CD4⁴ T cells expressing surface CD30 (29%) was seen along with increased mean fluorescence intensity. Both these results correlated with symptomatic disease severity score. Non-specific PHA stimulation failed to significantly affect CD30 expression. Conclusions There is a specific response to allergen in atopic asthma which causes significant increases in CD30 expression. This may correlate with disease severity.     

Similar potential to become activated and proliferate but differential kinetics and profiles of cytokine production of umbilical cord blood T cells and adult blood naive and memory T cells

Low alloreactivity of umbilical cord blood (UCB) T-cells may explain diminished graft-versus-host-disease after UCB transplantation. We investigated whether UCB T-cells have an intrinsic lower capacity to become activated. T-cells from UCB or adult blood (AB) were stimulated with anti-CD3 and anti-CD28 antibodies. On days 1-3 after stimulation, T-cell activation was determined by CD25 expression, proliferation was measured, and kinetics of cell division were analyzed by staining with CFSE. UCB and AB T cells exhibited similar numbers of activated and proliferating cells, but the extent of activation was lower in UCB T-cells. Enzyme-linked immunospot analysis showed lower levels and slower kinetics of IL-2, IL-4, IL-10, and IFN-gamma secreting cells for UCB T-cells. Comparison of UCB T-cells with CD45RA+ naive or CD45RO+ memory T cells purified from AB showed relatively low numbers of IL-4 and IL-10 secreting T cells in CD45RA+ AB T-cells and UCB T-cells as compared with CD45RO+ AB T cells. Numbers of IL-2 or IFN-gamma secreting cells in adult CD45RA+ T-cells were lower than in CD45RO+ T-cells but higher than in UCB T-cells. Thus diminished reactivity of UCB T-cells was not caused by a lower capacity to become activated and proliferate but may be explained by a lower extent of activation in UCB T cells, the absence of memory T cells in UCB, and differences between naive T cells from UCB and AB.     

Differential expression of the cytokine receptors for human interleukin (IL)-12 and IL-18 on lymphocytes of both CD45RA and CD45RO phenotype from tonsils, cord and adult peripheral blood

The objective of this study was to demonstrate the variable expression of cytokine receptors on naive versus memory human CD4+ T cell subpopulations in tonsillar tissue, cord blood and adult blood. We prove that the receptors for both interleukin (IL)-12 and IL-18 are expressed exclusively on memory T cells. This observation was seen not only on the CD45RO+ memory T cells but also on a significant percentage of the CD45RA+, CD62L-, CD27- and CCR7- populations. Furthermore, CD45RA+ CD62L+, CD27+ or CCR7+ CD4+ T cells that expressed IL-12Rbeta1 and IL-18Ralpha did not express CD31, a marker for recent thymic emigrants. We reveal that cord blood lymphocytes do not express IL-12Rbeta1 whereas IL-18Ralpha expression was detected at low levels. Importantly, the IL-12Rbeta2 signalling chain, which is absent in all resting T cells, was up-regulated in both CD45RA+ and CD45RO+ T cells as a result of stimulation with anti-CD3 and anti-CD28 in vitro. This observed up-regulation was, however, restricted to 80% of the total CD4+ population. Finally, a very small proportion of the CD4+ CD45RO+ tonsillar T cells expressed the IL-12 and IL-18 receptors, thereby establishing the differential expression of these receptors between peripheral and tonsillar memory T cell subpopulations.     

Dendritic cell number is related to IL-4 expression in the airways of atopic asthmatic subjects

BACKGROUND: Airway dendritic cells are essential for stimulating naive T cells in response to inhaled antigen and for the development of allergic sensitization. IL-4 in vitro can distinguish dendritic cell lines from peripheral blood mononuclear cells. Our study had the following aims: 1) to compare the distribution of CD1a+ dendritic cells and IL-4+ cells, in the bronchial mucosa of asthmatics and controls 2) to determine the relationship between the numbers of CD1a+ dendritic cells and IL-4+ cells in the bronchial mucosa of asthmatics 3) to determine whether CD1a+ cells express the IL-4 receptor. METHODS: Twenty atopic asthmatic and eight normal subjects were studied. In each subject, bronchoscopy with bronchial biopsies was performed. CD1a, IL-4, and IL-4 receptor expressions were evaluated by immunohistochemistry. RESULTS: The number of CD1a+ and IL-4+ cells was significantly higher in asthmatics than controls. The number of CD1a+ cells was positively correlated to the number of IL-4 + cells. Bronchial biopsy serial section studies showed that CD1a+ cells express the receptor for IL-4. CONCLUSIONS: These results suggest that an increased amount of IL-4 may play a physiopathologic role in maintaining the dendritic cell pool in vivo. Therefore, because of possible IL-4 activity on antigen-presenting cells in T-cell immune responses to allergens, an important new role of IL-4 in asthma inflammation can be envisaged.     

Relationship between dendritic cells and activated eosinophils in induced sputum of asthmatics

It has been suggested that dendritic cells (DCs) are critical antigen presenting cells for eosinophilic airway inflammation in a mouse model of asthma, and cysteinyl leukotrienes may play a role in DC trafficking in asthmatics. We investigated whether the number of DCs is increased in the induced sputum of both atopic and nonatopic asthmatics and is related to activated eosinophil count in the sputum. Sputum was induced by inhalation of hypertonic saline in 9 atopic and 12 nonatopic asthmatics and 10 nonatopic normal controls, and differential cell counts were performed. DCs and activated eosinophils were identified by immunocytochemistry with monoclonal antibodies (anti-CD1a and EG2, respectively). There were significantly higher percentages of eosinophils, EG2+ cells, and CD1a+ DC in the sputum of atopic and nonatopic asthmatics compared with normal controls, respectively. In asthmatics, the percentage of CD1a+ DC was significantly correlated with that of EG2+ cells (Rs=0.62, p=0.004). We demonstrated that the increased number of DCs was evident in the induced sputum of both atopic and nonatopic asthmatics, and the DC number was related to the activated eosinophil count, which suggests that DCs may contribute to the ongoing eosinophilic inflammation in asthmatic airways, and vice versa.     

Effect of house dust mite immunotherapy on transforming growth factor beta1-producing T cells in asthmatic children.

BACKGROUND: Recent evidence suggests that regulatory T cells (Treg cells) and immunosuppressive cytokines, such as transforming growth factor BETA1 (TGF-BETA1) and interleukin 10 (IL-10), may have a role in clinically effective allergen specific immunotherapy (SIT). OBJECTIVE: To evaluate the effect of SIT on the induction of Treg cells in house dust mite-allergic children and on the expression of specific Treg cell markers (cytotoxic T-lymphocyte-associated protein 4 [CTLA-4], IL-10, and TGF-BETA1). METHODS: In this uncontrolled open-label study, the percentage of peripheral blood CD4+ Treg cells (CD69 CD45RO+CTLA-4+ and CD3+CD4+CD25+FOXP3+) and the expression of molecules associated with their functions (CTLA-4, TGF-BETA1, and IL-10) were analyzed using flow cytometry in 16 children allergic to house dust mites before and at 3 and 12 months of subcutaneous SIT. Clinical variables, such as symptom score, medication requirements, forced expiratory volume in 1 second, peak expiratory flow rate, and serum IgE levels, were also determined. Ten healthy children were included as controls. RESULTS: All the clinical variables improved during immunotherapy. The percentage of CD4+CD25+CD69-CD45RO+ Treg cells remained unchanged. The percentage of CTLA-4+ -expressing Treg cells transiently increased after 3 months of immunotherapy, whereas the percentage of FOXP3+ Treg cells did not change after 1 year of immunotherapy. Levels of IL-10+ cells transiently decreased after 3 months of immunotherapy. Four children who required inhaled fluticasone propionate administration for significant symptom worsening had no statistically significant increase in TGF-BETA1-secreting T cells at 12 months of SIT, in contrast to 12 children without inhaled corticosteroid treatment. CONCLUSIONS: The increase in TGF-BETA1-positive T cells only in children without significant symptom worsening requiring inhaled corticosteroid treatment limits the usefulness of TGF-BETA1 in monitoring response to allergen immunotherapy.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo.

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

Th1/Th2 profile in peripheral blood in atopic cough and atopic asthma

BACKGROUND: Eosinophilic tracheobronchitis with cough hypersensitivity, abbreviated as atopic cough, is an important cause of chronic cough. The reason for the absence of airway hyper-responsiveness is unknown, differing from asthma, a Th2 cytokine-mediated disorder. OBJECTIVE: To compare the type 1 helper T cell (Th1)/Th2 balance in the peripheral blood from subjects with atopic cough and atopic asthma, we assessed the intracellular cytokine production at the single-cell level. METHODS: Thirty-six subjects (10 patients with atopic cough, 18 with atopic asthma, and eight control subjects) were included. Intracellular IL-4 and IFN-gamma were detected in CD4+ T cells by flow cytometry. RESULTS: A significantly lower ratio of IFN-gamma-/IL-4-producing CD4+ T cells after phorbol 12-myristate acetate/ionomycin stimulation was found in patients with atopic cough and atopic asthma compared with normal subjects. In comparison between atopic patients, the ratio of IFN-gamma-/IL-4-producing cells was significantly higher in atopic cough than in atopic asthma. However, the proportion of IL-4-producing CD4+ T cells was significantly higher in patients with atopic asthma than in normal control subjects and no significant difference was detected between patients with atopic cough and normal subjects. No significant difference in the proportion of IFN-gamma-producing cells was found between the subjects. Overall, the total IgE levels were positively correlated to the IL-4-producing cells and inversely correlated to the ratio of IFN-gamma-/IL-4-producing cells. CONCLUSION: These results show the lower degree of Th2 cytokine predominance in atopic cough compared with atopic asthma and suggest the relation between the Th1/Th2 balance and atopic status.     

Age-related defects in CD2 receptor-induced activation in human T-cell subsets.

It is well documented that the proliferative capacity of T cells declines with advancing age. There are, however, conflicting data as to the role of the accessory cell and whether or not this loss in responsiveness extends to all T-cell stimuli and to all T cells. We report here on the capacity of subpopulations of peripheral blood CD4+ T cells from the healthy aged to proliferate in response to anti-CD2 receptor-induced activation in the complete absence of accessory cells by using various exogenous cofactors as second signals. These costimulatory factors included phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-1, IL-2, IL-6 and IL-7 and the monoclonal antibodies, anti-CD28 and anti-CD44. Under these conditions, the proliferative responsiveness of CD4+CD45RO+ T cells from the aged was found to be comparable to young control cells for all stimuli tested, except anti-CD2 plus IL-7. This suggests that signal transduction pathways involving CD2, except IL-7-mediated events, are essentially intact in 'old' memory CD4+ T cells. On the other hand, several cofactors, namely IL-2, IL-6, IL-7 and to a lesser extent IL-1 beta and PMA, failed to support adequately CD2-induced activation in 'old' CD4+CD45RA+ T cells suggesting severe and multiple signalling deficiencies in this subset.     

Immunity of fungal infections alleviated graft reject in liver transplantation compared with non-fungus recipients

Objective: To evaluate of the immune tolerance in adult LT recipients with Invasive fungal infections (IFIs). Methods: 109 consecutive LT recipients who received LT were included. Percentage of T subsets (CD4+CD25hiCD127- T cells, CD4+CD25loCD45RA+ T cells, CD4+CD25loCD45RA- and CD4+CD45RA-CD45RO+ T cells populations), levels of cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, IL-12p70, IL-17, TNF-α, TNF-β and GM-CSF) were detected by FACS and Bioplex in peripheral blood. Biopsy specimens were fixed, monoclonal antibodies against CD4, Foxp3 and IL-17 were applied to the above sections and FISH was performed. Results: The risk of acute rejection was decreased in fungal infected liver transplant recipients comparing with non-fungal infected group. CD4+CD25hiCD127T cell population was increased in peripheral blood and memory CD4+CD45RA-CD45RO+ T cell population decreased. There was significant lower levels observed in naïve CD4+CD25loCD45RA+ and CD4+CD25loCD45RA- T cell populations in fungal infected liver transplant. Moreover, IL-2, IL-6, IL-10 and GM-CSF were decreased. However, no significant difference with IL-4 and IL-8 in serum in two infected LT recipients. Conclusion: The incidence of graft rejection in liver transplantation recipients with fungal infections was lower than the non-fungal group. It is important to assess the risk during pretransplant and postoperation for liver transplantation.     

Effect of inhaled interleukin-5 on eosinophil progenitors in the bronchi and bone marrow of asthmatic and non-asthmatic volunteers

BACKGROUND: Asthma is characterized by increases in mature eosinophils and their progenitors within the bronchus and bone marrow. IL-5 plays a key role in eosinophil development in the bone marrow and at the site of allergic inflammation. We therefore studied the effects of nebulized IL-5 on eosinophils, their progenitors and in situ haemopoiesis within the airway and bone marrow. METHODS: Nine atopic asthmatics and 10 non-atopic non-asthmatic control volunteers inhaled 10 microg of IL-5 or placebo via a nebulizer in a double-blind, randomized, cross-over study. Bronchoscopy, bone marrow aspiration and peripheral blood sampling were performed 24 h after nebulization. Four weeks later, volunteers inhaled the alternative solution and underwent a repeat bronchoscopy and bone marrow aspiration. RESULTS: Inhalation of IL-5 significantly decreased CD34(+)/IL-5Ralpha mRNA(+) cells within the bronchial mucosa and the percentage of CD34(+) cells that were CCR3(+) within the bone marrow of atopic asthmatic, but not control, volunteers. Inhalation of IL-5 also induced a significant increase in bronchial mucosal eosinophils in the non-atopic non-asthmatic control volunteers, but not in the asthmatics. IL-5 had no effect on spirometry or airways hyper-reactivity in either group. CONCLUSIONS: Inhaled IL-5 modulated eosinophil progenitor numbers in both the airways and bone marrow of asthmatics and induced local eosinophilia in non-asthmatics.     

Different profiles of T-cell IFN-gamma and IL-12 in allergen-induced early and dual responders with asthma

BACKGROUND: IFN-gamma and IL-12 are anti-inflammatory cytokines released from various cells, including T cells. Allergen inhalation by atopic subjects with asthma results in 2 bronchoconstrictor phenotypes, termed isolated early and dual responders . Persistence of allergen-induced airway response and inflammation is a distinctive feature of dual responders. OBJECTIVE: To evaluate the roles of IFN-gamma and IL-12 in resolving allergen-induced airway inflammation by comparing T lymphocytes (CD4 + and CD8 + cells) producing these cytokines in isolated early and dual responders. METHODS: Twenty-four subjects with asthma (12 isolated early and 12 dual responders) were challenged with inhaled allergen. Peripheral blood and induced sputum were taken before and 1 day, 3 days, and 7 days after challenge. Frequency of IFN-gamma, IL-12, IL-4, and IL-13 producing CD4 + and CD8 + cells was assessed by using flow cytometry. RESULTS: After allergen, both CD4 + and CD8 + IFN-gamma positive cells in peripheral blood significantly decreased in dual responders only, whereas CD4 + and CD8 + IFN-gamma positive cells in induced sputum significantly increased in isolated early responders only. By contrast, IL-12 positive cells in peripheral blood significantly increased after allergen challenge only in isolated early responders. The ratio of CD4 + and CD8 + IL-4/IFN-gamma positive cells in peripheral blood significantly decreased in isolated early responders by 3 days and had recovered by 7 days. CONCLUSION: These results suggest that contrasting profiles of IFN-gamma and IL-12 production may be responsible for different time courses of allergen-induced airway responses between isolated early and dual responders.     

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.     

Induction of IL-10+CD4+CD25+ T cells by grass pollen immunotherapy

BACKGROUND: Immunotherapy involves the modulation of allergen-specific T-cell responses, either T(H)2-to-T(H)1 immune deviation or, in bee venom-treated patients, induction of IL-10 production by CD4+CD25+ T cells. IL-10-producing CD4+CD25+ regulatory T cells have emerged as potential mediators of immune tolerance in numerous murine models of immunopathology. OBJECTIVE: The aim of this study was to evaluate the role of IL-10 production and CD4+CD25+ T cells in the response to grass pollen immunotherapy. METHODS: PBMCs were isolated from patients after 1 year of grass pollen immunotherapy and from matched untreated atopic and healthy control subjects. After 6 days of in vitro stimulation with Phleum pratense, production of IL-10, IL-5, IL-4, and IFN-gamma and proliferation and numbers of CD4+CD25+ T cells were measured. T cells were then stimulated for a further 5 hours with phorbol 12-myristate 13-acetate and ionomycin and assessed for intracellular IL-10 by means of flow cytometry. RESULTS: Patients undergoing immunotherapy produced significantly more IL-10 than atopic control subjects (patients undergoing immunotherapy, 116 +/- 21 pg/mL [n = 11]; atopic patients, 30 +/- 5 pg/mL [n = 11]; P <.001), and the number of CD4+CD25+ cells identified after allergen stimulation was also greater in the immunotherapy group. The numbers of CD4+CD25+ T cells correlated positively with activation as measured by proliferation in both of the control groups but not in the immunotherapy group. Moreover, only T cells from patients undergoing immunotherapy were positive for intracellular IL-10, and these were almost exclusively CD4+CD25+ cells. CONCLUSION: Grass pollen immunotherapy results in a population of circulating T cells that express the IL-10(+) CD4+CD25+ phenotype in response to allergen restimulation.     

Cytokine production by cord blood mononuclear cells stimulated with cows milk proteins in vitro: interleukin-4 and transforming growth factor β-secreting cells detected in the CD45RO T cell population in children of atopic mothers

BACKGROUND: Food antigens from the maternal circulation may sensitize fetal T cells in utero and be an important determinant in the development of food allergy. METHODS: Here we have examined the spontaneous and recall response to cow's milk proteins of cord blood mononuclear cells (CBMC) of newborn children, using single cell ELISPOT assays. RESULTS: In term newborns, confirming previous studies, the spontaneous cytokine response of CBMC is dominated by IL-4, IL-5, IL-10, and as shown here for the first time, TGF-beta. For TGF-beta only, the response of samples from infants of atopic mothers was significantly lower than samples from infants of non-atopic mothers. In vitro stimulation of CBMC with bovine serum albumin, casein and beta-lactoglobulin resulted in a significant increase of all cytokine-secreting cells, again dominated by T helper type 2 (Th2) cytokines. There was a clear tendency for samples from infants of atopic mothers to have lower Th2 responses than samples from infants of non-atopic mothers, which was particularly significant for both IL-4 and TGF-beta. Spontaneous cytokine secreting cells were virtually absent in cord blood from infants < 34 weeks gestation, as were cows milk protein-induced responses, although they were readily detectable in samples from infants aged > 34 weeks. To explore whether the cytokine secreting cells were in the naive CD4+ CD45RA population or memory CD4+ CD45RO T cells, these subsets were purified by positive and negative selection and tested for spontaneous and cows milk protein-induced cytokine responses. Strikingly, although the responses were small, the CD45RO+ cells from children of atopic mothers showed significant spontaneous and antigen-specific IL-4 and TGF-beta responses, whereas the same population from infants of non-atopic mothers showed virtually no response. In addition CD45RA+ cells from infants of mothers with maternal atopy showed decreased IL-4 and TGF-beta responses, especially the latter. CONCLUSIONS: The cows milk antigen-specific IL-4 and TGF-beta responses preferentially seen in the memory cell subset of infants with a maternal history of atopy strongly suggests Th2 skewing to dietary antigens in utero.     

Regulation of allergic inflammation by skin-homing T cells in allergic eczema

BACKGROUND: In allergic inflammations of the skin, the pivotal role of CD45RO+ (memory/effector) T cells expressing the cutaneous lymphocyte-associated antigen (CLA) was demonstrated. In both atopic dermatitis (AD) and contact dermatitis (CD), T cells specific to skin-related allergens were confined to the CLA+ T cell population. Our research was aimed to further characterize these T cells in AD. METHODS: CD4+ and CD8+ subsets of CLA+ CD45RO+ T cells were purified from peripheral blood of AD patients and healthy control individuals. We studied, in vivo activation patterns, cytokine profiles, immunoglobulin isotype regulation and the influence of these cells on eosinophil survival and apoptosis. RESULTS: The CLA+ CD45RO+ T cells represent an in- vivo-activated memory/effector T cell subset as shown by surface expression of activation markers, spontaneous proliferation and a lower activation threshold via TCR/CD3 triggering. These cells contain and spontaneously release high amounts of preformed IL-5 and IL-13 but only very little IL-4 and IFN-gamma in their cytoplasm, as demonstrated by intracellular cytokine staining immediately after purification. Moreover, CLA+ memory/ effector T cells induce IgE production in B cells and enhance eosinophil survival by inhibiting eosinophil apoptosis in AD. In comparison, the CLA- population represents a resting memory T cell fraction, induces rather IgG4 in B cells and does not show any effect on eosinophil survival and apoptosis. CONCLUSION: Our results indicate that in-vivo-activated both CD4+ and CD8+ memory/effector T cells with skin-homing property play a specific and decisive role in the pathogenesis and exacerbation of AD. In contrast, resting memory T cells of atopic individuals retain normal, nonallergic immune functions.