Induction of T ''regulatory'' cells by standardized house dust mite immunotherapy: an increase in CD4+CD25+ interleukin-10+ T cells expressing peripheral tissue trafficking markers

BACKGROUND: Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs. OBJECTIVE: This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT. METHODS: A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-gamma and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation. RESULTS: At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P < 0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P < 0.05) compared with baseline. CD4+ and CD8+ T cell IFN-gamma production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L(-), CD4+CD49d(hi), CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P < 0.05). IL-10 staining co-localized with CD4+CD25+ T cells. CONCLUSION: Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.     

Defective suppression of Th2 cytokines by CD4+CD25+ regulatory T cells in birch allergics during birch pollen season

BACKGROUND: CD4(+)CD25+ regulatory T cells suppress proliferation and cytokine production by human T cells both to self-antigens and exogenous antigens. Absence of these cells in human newborns leads to multiple autoimmune and inflammatory disorders together with elevated IgE levels. However, their role in human allergic disease is still unclear. OBJECTIVE: This study aimed to evaluate the capacity of CD4(+)CD25+ regulatory T cells to suppress proliferation and cytokine production outside and during birch-pollen season in birch-allergic patients relative to non-allergic controls. METHODS: CD4+ cells were obtained from blood of 13 birch-allergic patients and six non-allergic controls outside pollen season and from 10 birch-allergic patients and 10 non-allergic controls during birch-pollen season. CD25+ and CD25- fractions were purified with magnetic beads and cell fractions, alone or together in various ratios, were cultured with antigen-presenting cells and birch-pollen extract or anti-CD3 antibody. Proliferation and levels of IFN-gamma, IL-13, IL-5 and IL-10 were measured by thymidin incorporation and ELISA, respectively. Numbers of CD25+ cells were analysed by flow cytometry. RESULTS: CD4(+)CD25+ regulatory T cells from both allergics and non-allergics potently suppressed T cell proliferation to birch allergen both outside and during birch-pollen season. However, during season CD4(+)CD25+ regulatory T cells from allergic patients but not from non-allergic controls were defective in down-regulating birch pollen induced IL-13 and IL-5 production, while their capacity to suppress IFN-gamma production was retained. In contrast, outside pollen season the regulatory cells of both allergics and non-allergic controls were able to inhibit T-helper 2 cytokine production. CONCLUSION: This is the first study to show differential suppression of Th1 and Th2 cytokines, with CD4(+)CD25+ regulatory T cells from birch-pollen-allergic patients being unable to down-regulate Th2, but not Th1 responses during birch-pollen season.     

Wasp venom immunotherapy induces a shift from IL-4-producing towards interferon-gamma-producing CD4+ and CD8+ T lymphocytes

BACKGROUND: Venom immunotherapy (VIT) has proven to be very effective in hymenoptera venom anaphylaxis. However, the underlying immunoregulatory mechanisms of venom immunotherapy remain poorly understood. Recent studies measured the total amount of cytokine in culture supernatans, suggesting a shift in cytokine production from Th2 to a Th1 cytokine profile during VIT. We wanted to examine the contribution of specific T lymphocyte subpopulations, which is impossible using an extracellular method to determine cytokines in supernatants. OBJECTIVE: The present study was designed to evaluate the effect of VIT on the percentages of type 1 (IL-2, interferon-gamma (IFN-gamma)) and type 2 (IL-4) CD4+ and CD8+ T lymphocytes of patients with wasp venom anaphylaxis during immunotherapy. METHODS: Peripheral blood mononuclear cells of 20 individuals with a history of wasp sting anaphylaxis and a positive serum wasp venom specific IgE were isolated and in vitro stimulated with phorbol-12-myristate-13-acetate and ionomycin before VIT, at the end of a 5-day semirush VIT and at 6 months during VIT. Three-colour flow cytometric analysis was used for intracellular cytokine (IL-2, IFN-gamma, IL-4) detection in CD4+ (CD3+CD8-) T lymphocytes and CD8+ (CD3+CD8+) T lymphocytes. RESULTS: At the end of a 5-day semirush VIT, there was a significant decrease in percentage of IL-4-producing CD4+ and CD8+ T cells, compared with cytokine-producing cells before VIT (P = 0.0002 and 0.004). After 6 months of VIT, patients showed an increased number of IL-2-producing stimulated CD4+ and CD8+ lymphocytes compared with values before VIT (P = 0.002 and P = 0.0003). A higher amount of IFN-gamma-producing stimulated CD4+ and CD8+ cells was found after 6 months of VIT (P = 0.001 and P = 0.0006). There was no correlation between cytokine-producing cells and specific IgE for wasp. CONCLUSION: Venom immunotherapy induced a shift from IL-4-producing towards IFN-gamma-producing CD4+ as well as CD8+ T lymphocytes.     

Allergen-induced cytokine phenotypes in mice: role of CD4+ and CD8+ T cell populations

BACKGROUND: CD4+ T cells expressing type 2 cytokines have been implicated in the pathogenesis of asthma to high-molecular-weight allergens. Topical exposure of BALB/c strain mice to low-molecular-weight chemical contact and respiratory allergens stimulates type 1 and type 2 cytokine secretion phenotypes, respectively. OBJECTIVE: To examine the relative frequencies of cytokine-positive CD4+ and CD8+ T cells and their contributions to these cytokine secretion profiles. Methods Draining auricular lymph nodes were isolated 13 days after initiation of topical exposure of female BALB/c strain mice to chemical allergen, or to vehicle alone. The frequency of intracellular cytokine (IL-4 and IFN-gamma)-positive CD4+ and CD8+ lymphocytes was enumerated by flow cytometry. The relative contribution of CD4+ and CD8+ cells to cytokine secretion profiles was assessed by negative selection. RESULTS: Exposure to allergen resulted in an increased frequency of both IFN-gamma+ CD4+ and CD8+ lymphocytes, although there were no marked differences between trimellitic anhydride (TMA)- and 2,4-dinitrochlorobenzene (DNCB)-activated lymph node cells. Treatment with TMA induced approximately five times as many IL-4+ CD4+ cells as did exposure to DNCB. This pattern of cytokine staining was also observed for a further pair of contact and respiratory allergens; respectively, formalin and fluorescein isothiocyanate. CONCLUSION: These data demonstrate that the divergent immune responses induced in mice by different classes of chemical allergen are independent of changes in the frequency of IFN-gamma+ cells, but are associated with differential frequencies of IL-4-expressing CD4+ T cells.     

Peripheral blood CD4+ and CD8+ T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects

BACKGROUND: Increased production of IL-4 and IL-5 and decreased production of IFN-gamma by CD4+ T cells has been implicated in asthma pathogenesis. However, CD8+ T cells also produce type 1 and type 2 cytokines and the relative roles of CD4+ and CD8+ T cell cytokine production in asthma have not been previously studied. OBJECTIVE: To determine the production of the type 1 and type 2 cytokines by CD4+ and CD8+ T cell subsets in asthmatic and normal subjects. METHODS: Intracellular cytokine staining for IL-4, -5, -10, -13 and IFN-gamma was analysed in peripheral blood CD4+ and CD8+ T cells from 24 atopic asthmatic and 20 normal subjects. RESULTS: Both subsets of T cells produced all cytokines studied and there were no significant differences between CD4+ and CD8+ T cells in their capacity to produce either type 1 or type 2 cytokines. There were significantly increased frequencies of IFN-gamma-positive CD4+ (13.1 +/- 2.4%, vs. 7.3 +/- 1.4%) and CD8+ (20.0 +/- 2.9%, vs. 9.6 +/- 2.1%) T cells in asthmatic subjects compared with normal subjects (P < 0.05), but not in frequencies of CD4+ or CD8+ T cells staining positively for IL-4, -5, -10 or -13. CONCLUSION: The frequencies of peripheral blood CD8+ T cells producing type 1 and type 2 cytokines are comparable with the frequencies of CD4+ T cells. There was an increased frequency of IFN-gamma producing CD4+ and CD8+ T cells in asthmatic compared with normal subjects. Further studies investigating T cells derived from the airways and investigating various stages within the disease process are required to further elucidate the importance of type 2 and type 1 T cell cytokine production in the pathogenesis of human allergic disease.     

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.     

UCHL1+ (CD45RO+) ''memory'' T cells predominate in the CD4+ cellular infiltrate associated with allergen-induced late-phase skin reactions in atopic subjects.

We have previously shown that CD4+ T lymphocytes accumulated at the site of allergen induced late-phase reactions (LPR) in the skin of atopic subjects. In order to determine whether these were predominantly 'memory' or 'naive' cells, monoclonal antibodies recognizing isoforms of the CD45 common leucocyte antigen and immunocytochemical methods were used to study the composition of the T cell infiltrate. Allergen-induced late-phase skin reactions were biopsied 6, 24 or 48 h after allergen challenge. Memory (CD45RO+/UCHL1+) T cells predominated and few naive (CD45RA+/Leu18+) cells were identified. Double immunofluorescence was used to confirm that the UCHL1+ and Leu 18+ cells were CD4+ T lymphocytes. The selective recruitment of memory T cells to LPR sites is consistent with the active involvement of T lymphocytes in atopic allergic inflammation. A possible alternative explanation for apparently selective recruitment is the differential expression of endothelial adhesion molecules on memory and naive T lymphocytes.     

Characterization of chemokine receptor expression and cytokine production in circulating CD4+ T cells from patients with atopic dermatitis: up-regulation of C-C chemokine receptor 4 in atopic dermatitis

BACKGROUND: Th2 and Th1 cells have been suggested to express CCR3/CCR4 and CCR5/CXCR3, respectively. OBJECTIVE: We examined CCR3, CCR4, CCR5 and CXCR3 expression and cytokine production in peripheral blood CD4+ T cells from patients with atopic dermatitis (AD), which has been postulated to be a Th2-type cell-mediated disease, and then analysed the possible correlation between these values and the levels of several clinical parameters. METHODS: Intracellular cytokine production and chemokine receptor expression in peripheral blood CD4+ T cells from 40 AD patients and 20 sex- and age-matched healthy control subjects were studied by flow cytometry. RESULTS: The frequencies of IL-4- and IL-13-producing CD4+ T cells from patients with AD were significantly higher than those from healthy control subjects (IL-4:3.9 +/- 2.1% vs. 1.6 +/- 0.7%, P = 0.0005, IL-13:4.0 +/- 2.1% vs. 1.8 +/- 0.8%, P = 0.0023), whereas the frequencies of IL-2- and IFN-gamma-producing CD4+ T cells were significantly decreased in AD patients (IL-2:38.1 +/- 10.3% vs. 51.3 +/- 6.3%, P = 0.0003, IFN-gamma: 9.9 +/- 3.5% vs. 26.4 +/- 4.6%, P < 0.0001). The percentage of CCR4+ cells in CD4+ CD45RO+ T cells in AD patients was significantly higher than that in healthy control subjects (24.4 +/- 8.0% vs. 10.9 +/- 2.3%, P < 0.0001) and was correlated positively with the total serum IgE, serum lactic dehydrogenase (LDH) level, eosinophil number, eruption score, and IL-4 and IL-13 secretion in CD4+ T cells, and inversely with IL-2 and IFN-gamma secretion in CD4+ T cells. In contrast, CCR3 was not detected on circulating CD4+ T cells even in AD patients. On the other hand, the percentage of CCR5+ or CXCR3+ cells in CD4+ CD45RO+ T cells in AD patients was significantly decreased (CCR5:23.2 +/- 7.0% vs. 28.4 +/- 5.4%, P = 0.023, CXCR3:29.9 +/- 11.4% vs. 38.5 +/- 6.7%, P = 0.028) and was positively correlated with eruption score (P < 0.05). Multiple regression analyses showed that the percentage of CCR4 expression highly correlated with serum IgE, LDH, eosinophil number and eruption in AD patients. CONCLUSION: CCR4+ cells might be involved in the aetiopathogenesis of AD.     

The role of CD4+ T-cell subsets in determining transplantation rejection or tolerance

Summary: Peripheral tolerance to allogeneic organ grafts can be induced in rodents by treating with non-depleting CD4 and CD8 monoclonal antibodies. This tolerance is maintained by CD4⁺ T cells with a potent capacity to induce tolerance in further cohorts of T cells (i.e. infectious tolerance). We have cloned CD4⁺ T-cell subsets against the male transplantation antigen in vitro and find, in contrast to Th1 or Th2 clones that elicit rejection, that there is a distinct population of CD4⁺ T cells that suppress rejection by adoptive transfer (here called T reg ). In order to identify molecular markers associated with tolerance and gain insights into the mechanisms of action of T reg cells, we carried out serial analysis of gene expression. We identified genes overexpressed in T reg compared to Th1 and Th2 cultures and found that some of these correlated in vivo with CD4-induced transplantation tolerance rather than rejection. The genes overexpressed in T reg cultures and within tolerated skin grafts were primarily expressed by mast cells (e.g. tryptophan hydroxylase and FcεR1α), suggesting that regulatory cell activity and this form of tolerance may be associated with a localised but non-destructive form of Th2-like activation and a recruitment of mast cells.     

CD4+, CD8+, and CD4− CD8− T Cells in CSF and Blood of Patients with Multiple Sclerosis and Tension Headache

Two-colour flow cytometric analysis was performed on paired samples of peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with untreated multiple sclerosis (MS) and, for reference, subjects with muscular tension headache (TH) using anti-CD3, anti-CD4, anti-CD8, and anti-HLA-DR monoclonal antibodies in different combinations. CD4+/CD8+ T-cell ratio was increased in CSF compared to PB in both MS patients and TH subjects to a similar extent. This was mainly due to higher CD4+ T-cell levels in the CSF compartment. The proportion of HLA-DR+ T cells was higher in CSF than PB in both MS and TH; this increase of DR+ T cells in CSF was more prominent in MS. The level of CD4+ CD8+ T cells, which represent a subset of activated T cells, was not different between CSF and PB, either in MS or in TH. The proportion of CD4- CD8- T cells, which were found generally not to be blast cells, was lower in CSF compared to PB in both patient groups. However, their CSF level was higher and their PB level lower in MS compared to TH. Results point to an accumulation of activated T-helper cells in the CSF of both MS patients and healthy subjects. Fetal-type CD4- CD8- T cells bearing the unusual T-cell receptor gamma/delta seem to be selectively recruited to the CSF of MS patients.     

Multiple roles for CD4+ T cells in anti-tumor immune responses

Summary: Our understanding of the importance of CD4⁺ T cells in orchestrating immune responses has grown dramatically over the past decade. This lymphocyte family consists of diverse subsets ranging from interferon-γ (IFN-γ)-producing T-helper 1 (Th1) cells to transforming growth factor-β (TGF-β)-secreting T-regulatory cells, which have opposite roles in modulating immune responses to pathogens, tumor cells, and self-antigens. This review briefly addresses the various T-cell subsets within the CD4⁺ T-cell family and discusses recent research efforts aimed at elucidating the nature of the 'T-cell help' that has been shown to be essential for optimal immune function. Particular attention is paid to the role of Th cells in tumor immunotherapy. We review some of our own work in the field describing how CD4⁺ Th cells can enhance anti-tumor cytotoxic T-lymphocyte (CTL) responses by enhancing clonal expansion at the tumor site, preventing activation-induced cell death and functioning as antigen-presenting cells for CTLs to preferentially generate immune memory cells. These unconventional roles for Th lymphocytes, which require direct cell-to-cell communication with CTLs, are clear examples of how versatile these immunoregulatory cells are.     

CD137 ligand prevents the development of T-helper type 2 cell-mediated allergic asthma by interferon-γ-producing CD8+ T cells

BACKGROUND: Allergic asthma is a T-helper type 2 (Th2) cell-mediated chronic disease that is characterized by airway hyperreactivity (AHR) and chronic eosinophilic airway inflammation. Several studies suggest co-stimulatory molecules like CD137 as potential targets for therapeutic interventions in allergic airway disease. Recently, we could show in a murine asthma model that administration of an agonistic antibody against the receptor of the co-stimulatory molecule CD137 prevented and even reversed an already-established asthma phenotype. OBJECTIVE: The purpose of this study was to analyse the effect of stimulation of the CD137 ligand by a monoclonal antibody (CD137L mAb). METHODS: To induce an asthma-like phenotype, BALB/c mice were sensitized to ovalbumin (OVA), followed by an intrapulmonary allergen challenge. Anti-CD137L or control mAb were applied 1 day before OVA immunization or after the asthma phenotype was already established. RESULTS: Stimulation of the CD137L instead of the receptor by CD137L mAb prevents the development of an asthma-like phenotype but does not reverse established disease. While the receptor-mediated effect is partly mediated by anergy of CD4(+) T cells and partly by induction of IFN-gamma-producing CD8(+) T cells, the effect of the CD137L mAb is completely dependent on IFN-gamma-producing CD8(+) T cells: blockade of IFN-gamma and depletion of CD8(+) T cells fully abrogated the observed protective effect. In vitro experiments showed that the anti-CD137L mAb ligates directly to CD8(+) T cells and induces the generation of IFN-gamma by this cell population. CONCLUSION: Our results demonstrate that anti-CD137L mAb prevents disease development via IFN-gamma-producing CD8(+) T cells but is inferior to stimulation of the receptor that reverses established disease by a mechanism including CD4(+) T cell anergy.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

Natural CD4+ CD25+ regulatory T cells. Their role in the control of superantigen responses

Summary: CD4 regulatory T cells have a major role in controlling the immune response to self and foreign antigens. Natural CD4⁺ CD25⁺ T cells are a major component of the regulatory subset. Their absence is associated with the development of autoimmune and inflammatory diseases and with abnormal peripheral T-cell homeostasis. Two main characteristics discriminate natural CD4⁺ CD25⁺ T cells from their CD4⁺ CD25⁻ counterparts, namely their cytokine production profile and their behavior during tolerance induction. Natural CD4⁺ CD25⁺ T cells produce interleukin (IL)-10, a cytokine that contributes to their regulatory role. They do not produce IL-2 and are dependent on exogenous IL-2 for proliferation in vitro and in vivo . Studies of their response to superantigen administration in vivo show that they are resistant to clonal deletion but can be tolerized by anergy. Their resistance to apoptosis may contribute to their continuous regulatory function, as it allows them to maintain permanent control over effector T cells.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

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.     

The LFA-3 Adhesion Pathway is Differently Utilized by Superantigen-Activated Human CD4+ T-Cell Subsets

The superantigen SEA binds to MHC class II molecules and activates a large fraction of T cells as a result of interaction with particular TCR-V beta sequences. MHC class II transfected CHO cells induce a marginal CD4+ T-cell proliferation in the presence of SEA. CHO cells transfected with both MHC class II and LFA-3 (HLA-DR4/LFA-3 double transfectants) supported a vigorous T-cell proliferation and required 1000-fold lower SEA concentration than DR4-transfected cells. DR4/LFA-3 double transfectants presenting SEA to CD4+ T cells induced large amounts of IFN-gamma, while single DR4 transfectants failed to elicit IFN-gamma production. CD4+45RA+ naive T cells proliferated much more strongly compared with CD4+45R0+ memory T cells when SEA was presented by the DR4/LFA-3-transfected cells. In contrast, IFN-gamma production was only detected in CD4+45R0+ memory cells. The enhanced proliferation by the CD4+45RA+ naive T cells was not due to a stronger binding to the accessory DR4/LFA-3 cells. Human CD4+ T-cell lines mediated a low level of SEA-dependent cell-mediated cytotoxicity (SDCC) against DR4 target cells, whereas a strong SDCC was mediated against DR4/LFA-3-expressing target cells. These results demonstrate that superantigen-activated human CD4+ T cells require the adhesion molecule LFA-3 for optimal stimulation and that the CD4+ naive and memory T-helper cells are different in their response to LFA-3 as an accessory molecule.     

CD4+ memory T cells: functional differentiation and homeostasis

Summary: CD4⁺ T cells are central regulators of both humoral and cellular immune responses. There are many subsets of CD4⁺ T cells, the most prominent being T‐helper 1 (Th1), Th2, Th‐17, and regulatory T cells, specialized in regulating different aspects of immunity. Without participation by these CD4⁺ T‐cell subsets, B cells cannot undergo isotype switching to generate high‐affinity antibodies, the microbicidal activity of macrophages is reduced, the efficiency of CD8⁺ T‐cell responses and CD8⁺ T‐cell memory are compromised, and downregulation of effector responses is impaired. It therefore stands to reason that memory CD4⁺ T cells are likely to fulfill an important facilitator role in the maintenance and control of protective immune responses. This review discusses some issues of importance for the generation of memory CD4⁺ T cells and focuses in particular on their heterogeneity and plasticity, with respect to both phenotypic characteristics and function. Finally, we discuss a number of factors that affect long‐term maintenance of memory CD4⁺ T cells.     

CD4+CD25+ regulatory cells in acquired MHC tolerance

Summary: Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4⁺ cells. By analogy, regulatory CD4⁺ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4⁺ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4⁺-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8⁺ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-γ ELISPOT, and intracellular FACS analysis to study CD8⁺ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4⁺CD25⁺ cells that suppress the development of anti-donor CD8⁺ T-cell responses in vitro . We conclude that regulatory CD4⁺CD25⁺ cells initiate and/or maintain tolerance by preventing the development of CD8⁺ T-cell alloreactivity.