Dendritic cell phenotype in severe asthma reflects clinical responsiveness to glucocorticoids.

BACKGROUND: Subsets of patients with severe asthma remain symptomatic despite prolonged, high-dose glucocorticoid therapy. We hypothesized that the clinical glucocorticoid sensitivity of these asthmatics is reflected in differences in peripheral blood dendritic cell subsets. OBJECTIVE: To compare peripheral blood leucocyte populations using flow cytometry at baseline and after 2 weeks of systemic glucocorticoid (steroid) treatment to identify immunological differences between steroid-sensitive (SS) and steroid-resistant (SR) asthmatics. METHODS: Adult severe asthmatics (SS n = 12; SR n = 23) were assessed for their response to 2 weeks of therapy with oral prednisolone. Peripheral blood was obtained before and after therapy and stained for lymphocyte (CD3, CD19, CD4, CD8 and Foxp3) and dendritic cell markers (Lineage negative [CD3, CD14, CD16, CD19, CD20, CD56], HLA-DR+, CD304, CD11c, ILT3 and CD86). RESULTS: A higher median frequency of myeloid DCs (mDCs) but not plasmacytoid DCs (pDCs) was observed in the blood of SR as compared to SS asthmatics (P = .03). Glucocorticoid therapy significantly increased median B cell, but not T cell numbers in both cohorts, with a trend for increased numbers of Foxp3+ Tregs in SS (P = .07), but not SR subjects. Oral prednisolone therapy significantly reduced the median numbers and frequencies of total DCs and pDCs in both SS and SR asthmatics. Interestingly, the expression of HLA-DR and ILT3 was also reduced on pDCs in all patients. In contrast, therapy increased the median frequency of mDCs in SS, but reduced it in SR asthmatics. CONCLUSIONS: Myeloid DC frequency is elevated in SR compared with SS asthmatics, and mDC shows a differential response to oral prednisolone therapy.

Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease.

BACKGROUND: Associations 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). METHODS: In 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. RESULTS: Neonatal 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. CONCLUSION: Peri-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.

T cells producing the anti-inflammatory cytokine IL-10 regulate allergen-specific Th2 responses in human airways.

BACKGROUND: Murine models suggest a critical functional role for the anti-inflammatory cytokine IL-10 in local regulation of allergic airways inflammation. There is little corresponding information on human airway cells. This study aimed to investigate whether local IL-10 production regulates responses by respiratory mucosal leucocytes isolated from nasal polyps. MATERIALS AND METHODS: Nasal polyp tissue was harvested from 24 patients sensitised to aeroallergens with chronic rhinitis and polyposis undergoing routine polypectomy. Cells were isolated by matrix proteolysis. Cytokine production by polyp cells was determined by cytometric bead array (CBA) and intracellular cytokine analysis. Surface marker expression by polyp cells was determined by flow cytometry. RESULTS: Allergen stimulation significantly enhanced production of IL-10, but not IL-5 or IFN-γ by nasal polyp cell suspensions. Under the same conditions, neutralisation of IL-10 significantly increased allergen-specific IL-5 and IFN-γ production by nasal polyp cells. Cell depletion experiments showed that T cells themselves were primarily responsible for IL-10 production or for inducing its production by other cells. Intracellular cytokine staining confirmed production of IL-10 in the absence of IL-2 production by T cells in response to allergen. CONCLUSION: T cells within the human respiratory mucosa produce IL-10, which is capable of inhibiting pro-inflammatory Th2 and Th1 cytokine production in an antigen-specific fashion.

A randomized placebo-controlled trial of rush preseasonal depigmented polymerized grass pollen immunotherapy.

BACKGROUND: Specific subcutaneous immunotherapy (SCIT) for seasonal rhinoconjunctivitis with unmodified allergen extracts is effective, but limited by risk of side-effects and involves treatment over 3 years. We examined a depigmented polymerized grass pollen extract for immunogenicity and for clinical efficacy in a rush preseasonal regimen. METHODS: Depigmented polymerized grass pollen extract was tested for proliferation and cytokine production by peripheral blood mononuclear cells. A prospective, double-blind, placebo-controlled trial of 195 grass pollen allergic patients treated with preseasonal rush immunotherapy using depigmented polymerized allergenic extract of mixed grass pollen was performed over 2 years. Primary outcome was combined symptom and medication score (SMS) during the peak of the second grass pollen season. Secondary outcomes included combined score over the whole season, during the first grass pollen season, individual symptom and medication scores, quality of life, well days/hell days and responder analysis. Adverse events were classified using the EAACI scale. Grass pollen-specific IgE and IgG4 were measured before and during treatment. RESULTS: Depigmented polymerized extract stimulated dose-dependent T-cell proliferation and cytokine production. Patients treated with preseasonal SCIT showed improved combined scores during peak season at year 2 (median 3.93, interquartile range 0.77-6.27 vs median 5.86 for placebo, 3.11-8.36, P < 0.01). Most secondary outcomes were significantly better for active treatment. Side-effects were minimal, with no grade 3 or 4 reactions. CONCLUSIONS: Depigmented polymerized grass pollen extract is immunogenic and clinically effective in rush preseasonal SCIT. This form of immunotherapy may be an attractive option for some patients.

A novel technique to explore the functions of bronchial mucosal T cells in chronic obstructive pulmonary disease: application to cytotoxicity and cytokine immunoreactivity.

Bronchial mucosal CD8(+) cells are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but there are few data on their functional properties. We have developed a novel technique to outgrow these cells from COPD patients in sufficient numbers to examine effector functions. Endobronchial biopsies from 15 COPD smokers and 12 ex-smokers, 11 control smokers and 10 non-smokers were cultured with anti-CD3/interleukin (IL)-2 ± IL-15. Outgrown CD3(+) T cells were characterized in terms of phenotype (expression of CD4, 8, 25, 28, 69 and 56), cytotoxicity and expression of COPD-related cytokines. Compared with IL-2 alone, additional IL-15 increased the yield and viability of biopsy-derived CD3(+) T cells (12-16-day culture without restimulation) without alteration of CD4(+) /CD8(+) ratios or expression of accessory/activation molecules. Biopsy-derived T cells, principally CD8(+)/CD56(+) cells, exhibited statistically significantly greater cytotoxic activity in current or ex-smokers with COPD compared with controls (P < 0·01). Elevated percentages of CD8(+) T cells expressed interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-13 (P < 0·01) in current COPD smokers compared with all comparison groups. It is possible to perform functional studies on bronchial mucosal T cells in COPD. We demonstrate increased CD8(+)CD56(+) T cell cytotoxic activity and expression of remodelling cytokines in smokers who develop COPD.

Role of cysteinyl leukotrienes in human allergen-specific Th2 responses induced by granulocyte macrophage-colony stimulating factor.

BACKGROUND: The pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF), which is elevated in the lungs of atopic asthmatic patients, has been shown to enhance major histocompatibility class II expression of alveolar macrophages (AM). We hypothesized that exposure of AM and monocytes from atopic asthmatic patients to GM-CSF would enhance their antigen presenting function, and investigated putative mechanisms for this effect. METHODS: Alveolar macrophages were purified from bronchoalveolar lavage by plastic adherence. Monocytes and CD4(+) T cells were purified from peripheral blood by magnetic bead separation. Antigen-presenting cell (APC) were pretreated with GM-CSF, pulsed with allergen and cocultured with autologous T cells. T-cell proliferation was determined by tritiated thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay. RESULTS: Exposure of allergen-pulsed AM and peripheral blood monocytes to GM-CSF significantly increased allergen-specific T-cell proliferation and T helper 2 (Th2) cytokine production. The enhanced response was dependent on costimulation by CD86, but not CD80. Inhibition of the 5-lipoxygenase pathway abrogated GM-CSF-mediated upregulation by monocytes of allergen-specific interleukin-5 (IL-5) and IL-13 cytokine production. Blocking of the cysteinyl leukotriene receptor 1 (cysLT(1)) receptor by a specific receptor antagonist inhibited allergen-specific IL-5 production in response to GM-CSF pretreatment. CONCLUSION: Exposure to GM-CSF enhanced the capacity of human APC from atopic asthmatic patients to induce allergen-specific Th2 responses by a mechanism involving cysLT. Novel immunotherapies, targeting production of GM-CSF or its actions on APC have the potential, therefore, to prove beneficial in treatment of patients with inflammatory airway disease.

T regulatory cells and the control of allergic disease.

Allergic diseases are caused by the induction of T helper (Th)2 cells and IgE responses specific for common environmental antigens (allergens) in susceptible individuals. There is increasing interest in the role of both naturally occurring and induced regulatory T cell (Treg) populations in preventing these inappropriate immune responses and the underlying sensitisation to allergens. Current evidence suggests that Tregs may actively prevent Th2 responses to allergens occurring in healthy non-atopic individuals and that their function may be impaired in allergic patients. Evidence that existing therapies may act by modulating Treg function is reviewed. Future research aims to understand the mechanisms involved in the generation and function of allergen-specific Tregs. A primary aim is to promote the development of optimised therapeutic regimens with the capacity to provide long-lasting, allergen-specific, inhibitory mechanisms at the time and site of allergen challenge.

Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.

Allergic diseases are caused by aberrant T-helper-2 immune responses in susceptible individuals. Both naturally occurring CD4(+)CD25(+) regulatory T cells and inducible populations of antigen-specific interleukin-10-secreting regulatory T cells inhibit these inappropriate immune responses in experimental models. This article discusses the evidence that regulatory T-cell function might be impaired in allergic and asthmatic disease and that certain therapeutic regimens might function, at least in part, to promote regulatory T-cell generation. Current research strategies seek to exploit these observations to improve the generation of allergen-specific regulatory T-cell populations with the potential to provide the safe and long-term alleviation of disease symptoms.

Impaired secretion of interleukin-4 and interleukin-13 by allergen-specific T cells correlates with defective nuclear expression of NF-AT2 and jun B: relevance to immunotherapy.

BACKGROUND: Allergen immunotherapy (IT) is a successful treatment associated with decreased Th2 cytokine production by allergen-specific T cells. We have previously demonstrated (Faith et al., J Immunol 1997; 159:53-57) that inhibition of Th2 cytokine production in vitro correlates with impaired tyrosine kinase activity through the TCR. The transcription factor complex, nuclear factor of activated T cells (NF-AT), which regulates Th2 cytokine production is controlled by the activity of tyrosine kinases. OBJECTIVE: To address whether decreased Th2 cytokine production by allergen-specific CD4+ T cells following IT is correlated with altered translocation and nuclear expression of the NF-AT family member, NF-AT2, and the activator protein 1 (AP1) component of NF-AT, jun B. METHODS: T cell lines specific for insect venom phospholipase A2 (PLA) were derived from patients prior to and during conventional venom IT. Nuclear expressions of NF-AT and jun B were assessed following stimulation through the TCR. Th1 and Th2 cytokine and IL-10 production by insect venom-specific T cells were also determined. Results were compared with a well-established model system in which anergy was induced in cloned, allergen-specific Th2 cells. RESULTS: Impaired translocation and decreased expression of NF-AT2 and jun B were detected in PLA-specific T cell lines derived from bee venom-allergic individuals following 16 weeks treatment compared to pre-treatment. These results correlated with significantly reduced production of IL-4 and IL-13 and significantly increased production of IFN-gamma and IL-10 by PLA-specific T cells. Impaired IL-4 and IL-13 production also correlated with defective nuclear expression of NF-AT2/jun B in cloned, anergic allergen-specific Th2 cells. CONCLUSION: These results suggested that optimal production of IL-4 and IL-13 by allergen-specific T cells is dependent on the nuclear expression of NF-AT2 and jun B. Thus, specific inhibition of NF-AT2/jun B might be an option in novel and improved forms of allergen IT.

Glucocorticoids drive human CD8(+) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production.

Glucocorticoids are highly effective in the treatment of allergy and asthma and inhibit the synthesis of IL-4, IL-5 and IL-13 by disease-promoting CD4(+) Th2 cells. CD8(+) T cells also synthesize these cytokines, and the aim of this study was to investigate how glucocorticoids effect cytokine production by these cells. When CD8(+) T cells are stimulated with anti-CD3 and IL-2 plus IL-4 or dexamethasone, production of the anti-inflammatory cytokine IL-10 is low in both primary and secondary cultures restimulated with anti-CD3 and IL-2 alone. However, when both are present, a synergistic effect on IL-10 synthesis is observed. The additional presence of antigen-presenting cells (APC) in the priming culture maintains IL-10 levels, but inhibits IL-4 and IL-5 production. CD4(+) T cells develop a similar glucocorticoid-induced phenotype. These cells demonstrate regulatory activity and inhibit CD4(+) T cell activation in an IL-10-dependent manner. Earlier reports show glucocorticoids promote a Th2 phenotype by effects on purified naive T cells or pretreatment of APC. This study demonstrates, more critically, that when APC are present, glucocorticoids induce CD4 and CD8 T cell populations synthesizing high levels of IL-10, but greatly reduced amounts of disease-promoting IL-4 and IL-5.

Regulation of major histocompatibility complex class II antigens on human alveolar macrophages by granulocyte-macrophage colony-stimulating factor in the presence of glucocorticoids.

Alveolar macrophages (AM) present antigen poorly to CD4+ T cells and respond weakly to interferon-gamma (IFN-gamma) for up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecule expression. In atopic asthma, however, AM exhibit enhanced antigen-presenting cell (APC) activity. Since granulocyte-macrophage colony-stimulating factor (GM-CSF) is increased in the airways of asthmatic patients, we have investigated its role in modulating the APC function of AM. The effects of glucocorticoids were also studied since earlier studies showed optimal induction of MHC antigens on monocytes by GM-CSF in their presence. GM-CSF in the presence, but not the absence, of dexamethasone enhanced the expression of HLA-DR, -DP and -DQ antigens by AM. However AM and monocytes differed in the optimal concentration of steroid required to mediate this effect (10-10 m and 10-7 m, respectively). Induction of MHC antigens was glucocorticoid specific and independent of IFN-gamma. These studies suggest the existence of an IFN-gamma-independent pathway of macrophage activation, which may be important in regulating APC function within the lung.

Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

Dendritic cells from HIV-1-infected patients naturally express HIV-1 gp120 V3 loop-derived peptide ligands.

Little is known of the peptide ligands expressed in vivo on antigen-presenting cells (APC) or of the APC lineages involved. In this study we have addressed this question using HLA-DRbeta1*0101-restricted CD4 T cell clones (TLC) specific for a synthetic peptide based on the HIV-1 gp120 V3 loop consensus sequence for the Clade B isolates predominantly found in European and North American patients. These TLC were found to respond, in a dose-dependent manner, to freshly isolated HIV-infected patient APC in the absence of exogenously added peptides. Further APC purification showed that the naturally expressed peptide ligands were present in both the APC lineages shown to be infected with the virus and were most strongly detectable on purified blood dendritic cells. Peptides based on consensus sequences of viruses isolated from one of the patients over the period when naturally expressed peptide ligands could be detected were all found to stimulate TLC proliferation. These studies, therefore, show that peptide ligands derived from natural infection are detectable on APC lineages, particularly on dendritic cells which play an important role in the immune response to viruses. Even small differences in sequence between the vaccine isolate and the natural infection, if they occur in the key residues of protective T cell epitopes, could therefore have a profound effect on the efficacy of vaccines against viruses with high rates of mutation.

Leflunomide inhibits pyrimidine de novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans.

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic-acid dehydrogenase. Leflunomide (25 and 50 microM), like Brequinar (0.5 and 1 microM), a demonstrated dihydroorotic-acid dehydrogenase inhibitor, was cytostatic, not cytotoxic, with proliferation being halted in the G1 phase. Both drugs restricted the normal 4-8-fold mitogen-induced expansion of pyrimidine pools over 72 h to concentrations found in nonstimulated T-cells and [14C]bicarbonate incorporation into UTP, ATP, and GTP. Uridine (50 microM) restored expansion of all pools, but [14C]bicarbonate incorporation into ATP and GTP only, not UTP. [14C]Hypoxanthine salvage was also restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors. [14C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 microM Leflunomide. 100 microM Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidine-related effects reported for Leflunomide at 100 microM and above.

GM-CSF increases the ability of cultured macrophages to support autologous CD4+ T-cell proliferation in response to Dermatophagoides pteronyssinus and PPD antigen.

Previous studies have demonstrated an infiltration of monocytes and increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the asthmatic lung. To study the possible effects of this cytokine upon the differentiation and function of these newly recruited monocytes, we have developed a model in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of GM-CSF. After 7 days, the macrophages increased in size and granularity, had increased phagocytic activity, and expressed various adhesion molecules, CD14 and major histocompatibility complex (MHC) class II. The effects of GM-CSF on antigen presentation by cultured macrophages on the antigen-specific proliferative response of CD4+ T cells to Dermatophagoides pteronyssinus or purified protein derivative of tuberculin and the mitogen phytohaemagglutinin was determined. CD4+ T-cell proliferation was reduced when either antigen was presented by macrophages cultured in serum alone, compared with the values obtained with freshly isolated monocytes. However, CD4+ cell proliferation was comparable to that observed with monocytes when antigen was presented by macrophages which had been pre-cultured with 50 U/ml GM-CSF. CD4+ T-cell proliferation to phytohaemagglutinin was similar when all three populations were used as accessory cells. High numbers of macrophages partially suppressed CD4+ T-cell proliferation in response to antigen presented by monocytes, but there was no significant difference between macrophages cultured in the presence or absence of GM-CSF. This data suggests that GM-CSF directs monocyte differentiation into macrophages with an antigen-presenting, rather than a suppressive, phenotype. Elevated levels of GM-CSF in the asthmatic lung may therefore maintain recently recruited monocytes in an inflammatory and T-cell activating state.

A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection.

BACKGROUND: Genetic differences in immune responses may affect susceptibility to mycobacterial infection, but no specific genes have been implicated in humans. We studied four children who had an unexplained genetic susceptibility to mycobacterial infection and who appeared to have inherited the same recessive mutation from a common ancestor. METHODS: We used microsatellite analysis, immunofluorescence studies, and sequence analysis to study the affected patients, unaffected family members, and normal controls. RESULTS: A genome search using microsatellite markers identified a region on chromosome 6q in which the affected children were all homozygous for eight markers. The gene for interferon-gamma receptor 1 maps to this region. Immunofluorescence studies showed that the receptor was absent on leukocytes from the affected children. Sequence analysis of complementary DNA for the gene for interferon-gamma receptor 1 revealed a point mutation at nucleotide 395 that introduces a stop codon and results in a truncated protein that lacks the transmembrane and cytoplasmic domains. CONCLUSIONS: Four children with severe mycobacterial infections had a mutation in the gene for interferon-gamma receptor 1 that leads to the absence of receptors on cell surfaces and a functional defect in the up-regulation of tumor necrosis factor alpha by macrophages in response to interferon-gamma. The interferon-gamma pathway is important in the response to intracellular pathogens such as mycobacteria.

Neutrophils activated by granulocyte-macrophage colony-stimulating factor express receptors for interleukin-3 which mediate class II expression.

Freshly isolated peripheral blood neutrophils, unlike monocytes and eosinophils, do not bind interleukin-3 (IL-3) or respond to IL-3). We show that neutrophils cultured for 24 hours in granulocyte-macrophage colony-stimulating factor (GM-CSF) express mRNA for the IL-3 receptor (R) alpha subunit, as shown by RNase protection assays, and IL-3R alpha chain protein, as shown by cytometric analysis using two different specific monoclonal antibodies. This effect was selective for GM-CSF, because granulocyte colony-stimulating factor, tumor necrosis factor-alpha, interferon-gamma, and IL-1 failed to induce the IL-3 receptor. Saturation binding curves with 125I-IL-3 and Scatchard transformation showed the presence of about 100 high-affinity and 4,000 low-affinity receptors. Because neutrophils have been shown to express human leukocyte antigen (HLA)-DR in response to GM-CSF, we examined the possibility that IL-3 could augment HLA-DR expression on GM-CSF-treated cells. We found that neutrophils incubated with 30 ng/mL IL-3 as well as 0.1 ng/mL GM-CSF expressed a mean of 2.1-fold higher levels of HLA-DR than with GM-CSF alone (P < .005), confirming the signaling competence of the newly expressed IL-3R. This increase was seen even at maximal concentrations of GM-CSF and IL-3 can have an additive effect on mature human cells. The augmentation of HLA-DR by IL-3 was specific because it could be inhibited by a blocking anti-IL-3R antibody. Expression of class II molecules by neutrophils under these conditions may have significance for antigen presentation. These results provide further evidence for the role of GM-CSF as an amplification factor in inflammation by inducing neutrophil responsiveness to IL-3 produced by T cells or mast cells.

Inhibition of human T-cell responses to house dust mite allergens by a T-cell receptor peptide.

Recent analysis of the usage of T-cell receptor (TcR) beta chain variable region (V beta) gene elements by house dust mite (HDM)-reactive T cells from an atopic donor suggested that TcR-V beta 3 gene products may form a major component of the human T-cell repertoire reactive to this common allergen. In this study a peptide analog of the TcR-V beta 3 complementarity determining region 2 (CDR2) is shown to inhibit the polyclonal human T-cell response to HDM; this effect is specific because inhibition is dependent on the presence of V beta 3 + T cells. This experimental approach has been used to determine whether the pattern seen in T-cell clones derived from one atopic donor reflects TcR-V beta usage in the polyclonal response to allergen in the general population. Inhibition of more than 50% of the polyclonal response to allergen by V beta 3-CDR2 peptide was observed in 16 of 21 donors tested, suggesting that TcR-V beta 3 gene usage may form a major component of the human HDM repertoire and as such offer a suitable target for T cell-directed specific immunotherapy in HDM-allergic individuals. Depletion of CD8+ T cells abolishes peptide-mediated inhibition of CD4+ T-cell proliferation to HDM, suggesting that induction of a CD8+ regulatory T-cell subset by the CDR2 peptide may modulate HDM-specific allergic T-cell responses.