Tonsillar Cytokine Expression After Allergen and/or Virus Intervention
Tonsillar Cytokine Expression After Allergen and/or Virus Intervention
Hypotheses

 1. Immunotherapy induces tolerogenic effects to allergens in T cell regulation in tonsils.

 2. Influenza vaccination induces a strong interferon response and decreases Th2 response in
 tonsils.

 3. Influenza vaccination as an adjuvant on immunotherapy induces a better response to
 immunotherapy.
Background: Tonsils are the primary nasopharyngeal lymphoid tissue and constitute the first
 contact point of the immune system to allergens and infectious agents. A peripheral T cell
 repertoire exists in tonsils, and tonsils have an active role for inducing and maintaining
 peripheral tolerance to allergens. Intralymphatic immunotherapy is a promising alternative
 for a subcutaneous or sublingual immunotherapy. More data is needed on the interaction
 between the peripheral (tonsillar) T cell response and specific allergen exposure and impact
 of respiratory virus infections on tolerance induction. Objective: To study the effects of
 sublingual grass pollen immunotherapy and/or nasal live attenuated influenza vaccine on
 tonsillar T cell and interferon (IFN) expression. In addition, we collect material on the
 investigation of role of tissues in immune tolerance and chronicity in allergic diseases.

 Patients: Human tonsil samples will be obtained from less than 30-year-old routine
 tonsillectomy patients after sublingual grass immunotherapy (n=30), nasal live attenuated
 influenza vaccine (n=30 for atopic; and n=30 for non-atopic) and immunotherapy+influenza
 vaccine intervention (n=30). Controls without age limit or intervention (n=580) will also be
 recruited. Of these, age and atopy-matched controls will be selected as comparator groups for
 each intervention trial (n=30 for atopic; and n=30 for non-atopic). Control group will also
 be used for investigation of role of tissues in immune tolerance and chronicity in allergic
 diseases, immunomodulatory effects of respiratory viruses main focus being on human bocavirus
 and human rhinovirus infections, and in the search of new respiratory viruses.

 Design: Study intervention products will be administered openly but the immunology outcomes
 will be analysed blindly.

 Main outcomes: Gene expressions of IFN and T cell related genes will be the main outcomes and
 compared between intervention groups and correlated with the number of allergen extract doses
 or time from nasal influenza vaccination. Systemic immunologic outcomes (in peripheral blood
 mononuclear cells, blood RNA expression profiles, antibodies and cytokines in serum) will
 also be studied. The analyses include immunogenetics, virology, bacteriology and
 sensitization status. Clinical symptom scores will be recorded during the next 12 months from
 the screening visit.

 Anticipated results: We expect that our tonsil approach will offer a new in vivo model for
 the understanding immune tolerance induction and will give new insights to the immunologic
 mechanisms in lymphoid tissue level. We hope to demonstrate that the immunologic effects of
 sublingual pollen immunotherapy can be modified with a nasal live attenuated influenza
 vaccine and thereby it should give basis for other add-on treatments aiming for more
 effective immunotherapy. The influenza virus vaccine is expected to promote Th1 cell
 expression over Th2 cell expression. Since the immunologic outcomes will be correlated to
 clinical efficacy during next influenza and pollen seasons, we hope to understand the
 clinical significance of the immunologic outcomes.

 Innovation and impact: The unique strength of the project is to answer essential questions on
 development of allergen tolerance and a protective vaccine response in a human in vivo
 situation that cannot be answered in other settings. The proposed investigations may
 substantially enlarge our knowledge on the pathogenesis of organ directed allergic and
 non-allergic inflammation in humans, because the experiments will be performed directly with
 disease associated human cells and tissues. We expect that novel genes and pathways of immune
 regulation can be identified in tonsils by a detailed investigation of human immune response
 development to sublingual immunotherapy and influenza vaccine. The transcriptome and
 epigenome of these groups and further analyses of the role of T cells, B cells and innate
 lymphoid cells will provide essential information particularly on differences between atopic
 and non-atopic individuals, and anti-vaccine responses, natural viral infection, chronic
 viral infection, mechanisms of hypertrophic tonsillitis versus recurrent tonsillitis. The
 identification of new immune tolerance and immune activation pathways involved in the
 regulation of tissue cells is expected to provide novel knowledge for many other diseases in
 addition to asthma, because dysregulated immune system activation occurs in various human
 disorders such as cancer, autoimmunity, transplantation rejection and chronic infections.
Inclusion Criteria:

 -  elective tonsillectomy with or without adenotomy according to clinical indication

 -  age >4 and <30 years

 -  written informed consent from the study subject or his/her guardian

 -  Fluenz® will be used for ages >4 and <30 years, i.e. off-label use of ages >18 and <30
 years

 Exclusion Criteria:

 -  systemic anti-inflammatory medication within prior 4 weeks

 -  systemic diseases affecting the immune system e.g. autoimmune diseases, immune complex
 diseases or immune deficiency diseases other than allergy, asthma or atopic dermatitis

 -  malignancy, depression, psychiatric illness or medication; planned vaccination during
 the study period (vaccinations should not be given during study period)

 -  forced expiratory volume in 1 second (FEV1) is under 80% of normal value or asthma is
 in a bad balance for those patients who would participate in the immunotherapy

 -  sublingual grass pollen will not be given for children under the age of 5

 -  additional exclusion criteria for Grazax® include hypersensitivity to any of the
 excipients (gelatin [fish source], mannitol, sodium hydroxide), inflammatory
 conditions in the oral cavity with severe symptoms such as oral lichen planus with
 ulcerations or severe oral mycosis, patients with uncontrolled or severe asthma (in
 adults: FEV1 < 70% of predicted value after adequate pharmacologic treatment, in
 children: FEV1 < 80% of predicted value after adequate pharmacologic treatment)

 -  addition exclusion criteria for Fluenz® include hypersensitivity to the active
 substances, to any of the excipients (sucrose, dibasic potassium phosphate, monobasic
 potassium phosphate, gelatin [porcine, Type A], arginine hydrochloride, monosodium
 glutamate monohydrate, gentamicin [a possible trace residue], eggs or to egg proteins
 [e.g. ovalbumin] and children and adolescents younger than 18 years of age receiving
 salicylate therapy because of the association of Reye's syndrome with salicylates and
 wild-type influenza infection