Adrienne Verhoef

Grass pollen immunotherapy for hayfever is associated with increases in local nasal but not peripheral Th1 : Th2 cytokine ratios

Grass pollen immunotherapy is the only treatment for hayfever that is both effective and confers long‐term benefit. Immunotherapy may act by altering the local nasal mucosal T helper type 2 (Th2) to type 1 (Th1) cytokine balance either by down‐regulation and/or immune deviation of T‐lymphocyte responses. There is controversy as to whether these changes are detectable in peripheral blood. We therefore examined both local nasal and peripheral T‐cell responses to allergen exposure in the same subjects before and after immunotherapy. In a double‐blind trial of grass pollen immunotherapy, nasal biopsies were obtained at baseline and during the peak pollen season following 2 years of immunotherapy. Placebo‐treated patients showed a seasonal increase in CD3+ T cells (P = 0·02) and in interleukin‐5 (IL‐5) mRNA+ cells (P = 0·03) and no change in interferon‐γ (IFN‐γ ) mRNA+ cells (P = 0·2) in the nasal mucosa. In contrast, in the immunotherapy‐treated group, there were no changes in the number of CD3+ T cells (P = 0·3) and IL‐5 mRNA+ cells (P = 0·2) but a significant increase in the number of IFN‐γ mRNA+ cells (P = 0·03). Furthermore, clinical improvement in the immunotherapy‐treated group was accompanied by a seasonal increase in the ratio of IFN‐γ to IL‐5 mRNA+ cells in the nasal mucosa (P = 0·03). In contrast, there were no significant changes in peripheral T‐cell proliferative responses or cytokine production for IFN‐γ or IL‐5 in response to grass pollen either within or between the two treatment groups. We conclude that successful grass pollen immunotherapy was associated with an increase in the ratio of IFN‐γ to IL‐5 mRNA+ cells in the nasal mucosa, whereas these changes were not reflected by alterations in peripheral blood T‐cell proliferative responses or cytokine production before/after treatment.

Analysis of human T cell responses to the group II allergen of Dermatophagoides species: Localization of major antigenic sites

BACKGROUND IgE antibodies reactive with the group II allergens of Dermatophagoides species (house dust mite [HDM]) are a major component of the allergic immune response in HDM-allergic atopic individuals. METHODS Here we demonstrate, with the use of overlapping synthetic peptides of the group II allergen of Dermatophagoides pteronyssinus (Der p II), that polyclonal T cells isolated from the majority of atopic HDM-allergic individuals and healthy nonatopic control subjects respond to Der p II and that T-cell epitopes are present in all regions of the protein. RESULTS From comparison of peptide-specific T-cell proliferation in both groups of individuals, it appears that together peptides 61-86 and 78-104 are the most frequently recognized (16 of 18 individuals). We also observed that nine of the 18 individuals responded to T-cell epitopes in the region 11-50, and with Der p II-reactive T-cell clones, three distinct T-cell epitopes were mapped within the sequence 11-35. Also, with the use of T-cell clones, two additional epitopes were identified at residues 81-96 and 91-101. CONCLUSIONS These results suggest that T-cell epitopes located in these regions (11-50 and 61-104) are immunodominant. The value of this information in the potential application of Der p II peptides to desensitize HDM allergic responses is discussed.

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

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.

T cells from human allergen-induced late asthmatic responses express IL-12 receptor beta 2 subunit mRNA and respond to IL-12 in vitro.

IL-12 suppresses proallergic Th2-type cytokine production and induces Th1-type cytokine production by peripheral blood T cells from subjects with allergic disease. The objective of the present study was to examine the relevance of these findings to target organ T cell responses in human asthma. Bronchoalveolar lavage (BAL) and PBMC were collected from atopic asthmatics 24 h after fiberoptic allergen challenge of a segmental bronchus. BAL T cells and PBMC were cultured with allergen in the presence of recombinant IL-12 or IFN-γ, and cytokines were measured in culture supernatants after 6 days. IL-5 production by BAL T cells and PBMC was inhibited by IL-12 and, to a lesser extent, by IFN-γ. IL-12 also induced IFN-γ production by BAL T cells and PBMC. The effects of IL-12 nor IFN-γ on IL-5 production could not be reversed by neutralizing anti-IFN-γ or anti-IL-12 mAbs, respectively. Thus, the effect of neither IL-12 nor IFN-γ appeared to be mediated through induction of the other cytokine. In situ hybridization revealed that approximately one-third of BAL T cells expressed mRNA transcripts encoding the IL-12R β2 subunit following allergen challenge. Thus, human T cells obtained from BAL during asthmatic late responses, like T cells in the peripheral circulation, remain susceptible to immunomodulation by IL-12. These findings raise the possibility that IL-12 may hold therapeutic potential in allergic diseases such as asthma.

Threshold Signaling of Human Th0 Cells in Activation and Anergy: Modulation of Effector Function by Altered TCR Ligand

Molecular interactions between TCR and its natural ligand, in the presence of costimulatory signals, elicit T cell effector functions, whereas subtle changes in the structure of antigenic peptides may induce only selected T cell effector function including anergy. In this study, we have investigated the immunological activity of an altered TCR ligand (p 2, 28–40A34,36) derived from the immunodominant T cell epitope of the group 2 allergen of house dust mite, in which residues at positions 34 and 36 were substituted by alanine. Elevated IFN-γ synthesis was induced by equimolar concentrations of the analogue compared with native peptide (p 2, 28–40) and was paralleled by increased down-regulation of cell surface CD3. IL-5 and IL-10 production exhibit the same sensitivity to both peptides, implying that the induction of T cell effector functions are not all proportional to TCR occupancy. Both native peptide and the analogue bound to MHC class II (DRB1*1101) molecules with similar affinities. Furthermore, p 2, 28–40A34,36 induced T cell anergy at lower concentrations than native peptide. During the induction of anergy, TGF-β production was comparable for both peptides, whereas IL-10 secretion was markedly increased but more so in response to p 2, 28–40A34,36. Membrane expression of costimulatory ligands CD80 and CD86 was similar for native peptide and p 2, 28–40A34,36 and increased in activation, whereas only CD86 was elevated during anergy. The modulation of T cell effector function with altered TCR ligands may have practical applications in reprogramming allergic inflammatory responses through the induction of T cell anergy and/or the promotion of Th1 cytokines.

Clonal analysis of CD4 mediated accessory function on the effector activity of human CD4+ T cell subsets

Background: It has been reported for the peripheral T cell repertoire that CD4 molecules may enhance adhesion between T cells and antigen presenting cells and, through their physical association with T cell antigen receptors, contribute to signal transduction.

Production of human T-cell clones.

The study of monoclonal human T-cell populations has had a fundamental impact on our current knowledge of the function, specificity, and mechanisms of activation of these cells. The frequency of antigen-specific T cells in peripheral blood is low, necessitating several enrichment steps prior to the isolation of individual clones. Two different methods of limiting dilution cloning are described in this chapter, the choice of which depends on the availability of starting materials. After isolation, T-cell clones are expanded and then tested for specificity and cryopreserved, both of which are described.Recently, time-saving variations to the above methods have emerged. However, they require the use of sophisticated equipment and reagents, making them less economical than the established techniques.