Development and preliminary clinical evaluation of a peptide immunotherapy vaccine for cat allergy.

BACKGROUND: Allergic sensitization to cat allergens is common and represents a major risk factor for asthma. Specific immunotherapy (SIT) is effective but cumbersome and associated with IgE-dependent adverse events. Immunotherapy targeting allergen-specific T cells, with synthetic peptides representing T-cell epitopes, might improve safety and reduce the duration of treatment. OBJECTIVE: We sought to define major T-cell epitopes of Fel d 1 for peptide immunotherapy, generate a peptide vaccine, and evaluate its safety and tolerability in subjects with cat allergy. METHODS: We determined the binding affinities of Fel d 1 peptides for 10 commonly expressed HLA-DR molecules. Functionally immunodominant peptides were identified by means of proliferation and cytokine secretion. Histamine-releasing activity was assessed, and a peptide vaccine was formulated. Safety and tolerability were evaluated in a dose-ranging phase IIa clinical trial. RESULTS: MHC-binding sequences were identified throughout Fel d 1. Some regions contained multiple overlapping T-cell epitopes that bound multiple MHC molecules. Immunodominant sequences were identified on the basis of proliferative and cytokine (IFN-γ, IL-10, and IL-13) responses. Cat allergen extract, but not peptides, induced histamine release in blood basophils. A single administration of peptide vaccine was safe and well tolerated. The dose of vaccine resulting in the greatest inhibition of the late-phase skin response to intradermal whole allergen challenge was 3 nmol. CONCLUSIONS: Fel d 1 contains multiple overlapping MHC-binding motifs. A peptide vaccine comprising the immunodominant regions of the allergen was safe and well tolerated when given to subjects with cat allergy as a single dose. The dose of vaccine resulting in the greatest reduction in late-phase skin response was defined for future clinical development.

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.

T cell epitope immunotherapy induces a CD4+ T cell population with regulatory activity.

BACKGROUND: Synthetic peptides, representing CD4(+) T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils. METHODS AND FINDINGS: In this study we address the mechanism of action of peptide immunotherapy (PIT) in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs) after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4(+) cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4(neg) PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4(+) and CD8(+) PBMCs. CONCLUSION: This study provides evidence for the induction of a population of CD4(+) T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.

Secretion of IFN-gamma and not IL-2 by anergic human T cells correlates with assembly of an immature immune synapse.

We report differences in the supramolecular organization of the immunologic synapse (IS) formed by resting and anergic human T cells with agonist peptide-loaded antigen-presenting cells (APCs). T cells reactive to influenza A hemagglutinin peptide or Fel d 1 peptide 4 were rendered both anergic and regulatory by incubation with high doses of agonist peptide in the absence of APCs. At the IS between resting T cells and peptide-loaded APCs, both CD3epsilon and CD3zeta initially accumulate within a ring or arc before redistributing within 30 minutes to single or multiple foci more central to the contact. In contrast, at synapses formed by anergized T cells, CD3epsilon and CD3zeta remained organized within an arc or ring and failed to redistribute centrally. However, intercellular communication between anergic human T cells and agonist peptide-loaded APCs was not a null event, since it triggered secretion of T-cell interferon gamma (IFN-gamma) but not, for example, interleukin 2 (IL-2). Thus, distinct organizations of CD3 at the T-cell IS correlate with different cytokine profiles; the mature IS formed by resting T cells correlates with their production of both IFN-gamma and IL-2, whereas the immature IS formed by anergic T cells seems able to facilitate IFN-gamma but not IL-2 production.

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-gamma (IFN-gamma ) 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-gamma mRNA(+) cells (P = 0.03). Furthermore, clinical improvement in the immunotherapy-treated group was accompanied by a seasonal increase in the ratio of IFN-gamma 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-gamma 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-gamma 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.